Ciliated Epithelial Cell Differentiation at Air-Liquid Interface Using Commercially Available Culture Media
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.
KeywordsIn vitro models Cilia Nasal epithelial cells Mucociliary differentiation Air-liquid interface Nasal epithelium Multiciliated cells Primary ciliary dyskinesia Chronic obstructive pulmonary disease
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).
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