Skip to main content
SpringerLink
Log in
Book cover

Epidermal Cells pp 275–291Cite as

Ciliated Epithelial Cell Differentiation at Air-Liquid Interface Using Commercially Available Culture Media

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2109))

Abstract

The human nasal epithelium contains basal stem/progenitor cells that produce differentiated multiciliated and mucosecretory progeny. Basal epithelial cells can be expanded in cell culture and instructed to differentiate at an air-liquid interface using transwell membranes and differentiation media. For basal cell expansion, we have used 3T3-J2 co-culture in epithelial culture medium containing EGF, insulin, and a RHO-associated protein kinase (ROCK) inhibitor, Y-27632 (3T3 + Y). Here we describe our protocols for ciliated differentiation of these cultures at air-liquid interface and compare four commercially available differentiation media, across nine donor cell cultures (six healthy, two patients with chronic obstructive pulmonary disease (COPD), and one with primary ciliary dyskinesia (PCD)). Bright-field and immunofluorescence imaging suggested broad similarity between differentiation protocols. Subtle differences were seen in transepithelial electrical resistance (TEER), ciliary beat frequency, mucus production, and the extent to which basal cells are retained in differentiated cultures. Overall, the specific differentiation medium used in our air-liquid interface culture protocol was not a major determinant of ciliation, and our data suggest that the differentiation potential of basal cells at the outset is a more critical factor in air-liquid interface culture outcome. Detailed information on the constituents of the differentiation media was only available from one of the four manufacturers, a factor that may have profound implications in the interpretation of some research studies.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Fulcher ML, Randell SH (2013) Human nasal and tracheo-bronchial respiratory epithelial cell culture. Methods Mol Biol 945:109–121

    Article  Google Scholar 

  2. Danahay H, Pessotti AD, Coote J, Montgomery BE, Xia D, Wilson A, Yang H, Wang Z, Bevan L, Thomas C, Petit S, London A, LeMotte P, Doelemeyer A, Velez-Reyes GL, Bernasconi P, Fryer CJ, Edwards M, Capodieci P, Chen A, Hild M, Jaffe AB (2015) Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung. Cell Rep 10(2):239–252

    Article  CAS  Google Scholar 

  3. Hynds RE, Butler CR, Janes SM, Giangreco A (2016) Expansion of human airway basal stem cells and their differentiation as 3D Tracheospheres. Methods Mol Biol, vol 1576

    Google Scholar 

  4. Plasschaert LW, Zilionis R, Choo-Wing R, Savova V, Knehr J, Roma G, Klein AM, Jaffe AB (2018) A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature 560:377

    Article  CAS  Google Scholar 

  5. Garcia SR, Deprez M, Lebrigand K, Cavard A, Paquet A, Arguel MJ, Magnone V, Truchi M, Caballero I, Leroy S, Marquette CH, Marcet B, Barbry P, Zaragosi LE (2019) Single-cell RNA sequencing reveals novel cell differentiation dynamics during human airway epithelium regeneration. Development 146(20):dev177428

    Article  CAS  Google Scholar 

  6. Amatngalim GD, Schrumpf JA, Dishchekenian F, Mertens TCJ, Ninaber DK, van der Linden AC, Pilette C, Taube C, Hiemstra PS, van der Does AM (2018) Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence. Eur Respir J 51(4):1701009

    Article  Google Scholar 

  7. Li X (2016) In vitro toxicity testing of cigarette smoke based on the air-liquid interface exposure: a review. Toxicol In Vitro 36:105–113

    Article  CAS  Google Scholar 

  8. Smith CM, Kulkarni H, Radhakrishnan P, Rutman A, Bankart MJ, Williams G, Hirst RA, Easton AJ, Andrew PW, O'Callaghan C (2014) Ciliary dyskinesia is an early feature of respiratory syncytial virus infection. Eur Respir J 43(2):485–496

    Article  Google Scholar 

  9. Vries M, Bedke N, Smithers NP, Loxham M, Howarth PH, Nawijn MC, Davies DE (2016) Inhibition of Pim1 kinase, new therapeutic approach in virus-induced asthma exacerbations. Eur Respir J 47(3):783–791

    Article  Google Scholar 

  10. Faiz A, Weckmann M, Tasena H, Vermeulen CJ, Van den Berge M, Ten Hacken NHT, Halayko AJ, Ward JPT, Lee TH, Tjin G, Black JL, Haghi M, Xu CJ, King GG, Farah CS, Oliver BG, Heijink IH, Burgess JK (2018) Profiling of healthy and asthmatic airway smooth muscle cells following interleukin-1beta treatment: a novel role for CCL20 in chronic mucus hypersecretion. Eur Respir J 52(2)

    Article  Google Scholar 

  11. Mertens TCJ, Karmouty-Quintana H, Taube C, Hiemstra PS (2017) Use of airway epithelial cell culture to unravel the pathogenesis and study treatment in obstructive airway diseases. Pulm Pharmacol Ther 45:101–113

    Article  CAS  Google Scholar 

  12. de Courcey F, Zholos AV, Atherton-Watson H, Williams MT, Canning P, Danahay HL, Elborn JS, Ennis M (2012) Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology. Am J Physiol Cell Physiol 303(11):C1173–C1179

    Article  Google Scholar 

  13. Brewington JJ, Filbrandt ET, LaRosa FJ 3rd, Moncivaiz JD, Ostmann AJ, Strecker LM, Clancy JP (2018) Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies. JCI Insight 3(13)

    Google Scholar 

  14. Butler CR, Hynds RE, Gowers KH, Lee Ddo H, Brown JM, Crowley C, Teixeira VH, Smith CM, Urbani L, Hamilton NJ, Thakrar RM, Booth HL, Birchall MA, De Coppi P, Giangreco A, O’Callaghan C, Janes SM (2016) Rapid expansion of human epithelial stem cells suitable for airway tissue engineering. Am J Respir Crit Care Med 194(2):156–168

    Article  CAS  Google Scholar 

  15. Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, Timofeeva OA, Nealon C, Dakic A, Simic V, Haddad BR, Rhim JS, Dritschilo A, Riegel A, McBride A, Schlegel R (2012) ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol 180(2):599–607

    Article  CAS  Google Scholar 

  16. Reynolds SD, Rios C, Wesolowska-Andersen A, Zhuang Y, Pinter M, Happoldt C, Hill CL, Lallier SW, Cosgrove GP, Solomon GM, Nichols DP, Seibold MA (2016) Airway progenitor clone formation is enhanced by Y-27632-dependent changes in the Transcriptome. Am J Respir Cell Mol Biol 55(3):323–336

    Article  CAS  Google Scholar 

  17. Rayner RE, Makena P, Prasad GL, Cormet-Boyaka E (2019) Optimization of normal human bronchial epithelial (NHBE) cell 3D cultures for in vitro lung model studies. Sci Rep 9(1):500

    Article  Google Scholar 

  18. Smith CM, Djakow J, Free RC, Djakow P, Lonnen R, Williams G, Pohunek P, Hirst RA, Easton AJ, Andrew PW, O'Callaghan C (2012) ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software. Cilia 1:14

    Article  Google Scholar 

  19. Gowers KHC, Hynds RE, Thakrar RM, Carroll B, Birchall MA, Janes SM (2018) Optimized isolation and expansion of human airway epithelial basal cells from endobronchial biopsy samples. J Tissue Eng Regen Med 12(1):e313–e317

    Article  CAS  Google Scholar 

  20. Freestone PP, Hirst RA, Sandrini SM, Sharaff F, Fry H, Hyman S, O’Callaghan C (2012) Pseudomonas aeruginosa-catecholamine inotrope interactions: a contributory factor in the development of ventilator-associated pneumonia? Chest 142(5):1200–1210

    Article  Google Scholar 

  21. Lechner JF, LaVeck M (1985) A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J Tissue Cult Methods 9(2):43–48

    Article  Google Scholar 

Download references

Acknowledgments

We thank Dr. Gabriel Gata (Lonza), Lisa Schmidtke (PromoCell), Dr. Frankie Vanterpool (Thermo Fisher Scientific Scientific), and Dr. Angela Zhang (STEMCELL Technologies) for their feedback on this chapter and for assistance with establishing media composition. We are grateful to Prof. Sam Janes (University College London, U.K.) and Prof. Fiona Watt (Kings College London, U.K.) for providing the 3T3-J2 mouse embryonic fibroblasts used in our study. We also thank Dr. Kate Gowers (University College London, U.K.) and Dr. Kyren Lazarus (University College London, U.K.) for proofreading the manuscript.

This work was supported by the NIHR GOSH BRC and the Living Airway Biobank (C.O.). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. R.E.H. is supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust; WT209199/Z/17/Z).

Author information

Authors and Affiliations

  1. Respiratory, Critical Care & Anaesthesia, UCL Great Ormond Street Institute of Child Health, London, UK

    Dani Do Hyang Lee, Alina Petris & Christopher O’Callaghan

  2. UCL Respiratory, University College London, London, UK

    Robert E. Hynds

Authors
  1. Dani Do Hyang Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alina Petris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert E. Hynds
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher O’Callaghan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Robert E. Hynds or Christopher O’Callaghan .

Editor information

Editors and Affiliations

  1. Ottawa, ON, Canada

    Kursad Turksen

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media New York

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lee, D.D.H., Petris, A., Hynds, R.E., O’Callaghan, C. (2019). Ciliated Epithelial Cell Differentiation at Air-Liquid Interface Using Commercially Available Culture Media. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 2109. Humana, New York, NY. https://doi.org/10.1007/7651_2019_269

Download citation

  • DOI: https://doi.org/10.1007/7651_2019_269

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0250-8

  • Online ISBN: 978-1-0716-0251-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation