Table 2.

Comparison of three-dimensional organotypic airway epithelial culture methods.

	Respiratory—nasal and bronchial
	Airway explant spheroid	Airway basal cell spheroid	Long term expanding airway organoid
		Method 1—Dome Method 2–25% layer
Cultures
Cellular source	Freshly isolated non-dissociated airway epithelial sheets	Airway basal progenitor cells expanded as traditional primary cultures or conditionally reprogrammed cultures	Lung biopsies and cells from BAL fluid
Media	Standard, non-differentiating culture media e.g., BEGM	Differentiation media—same as those used for 2D ALI cultures	Media containing biochemical cues for self-renewal. Content: Advanced DMEM/F12 R-Spondin 1 Noggin FGF 7 FGF 10 A83-01 Y-27632 SB202190 B27 supplement N-Acetylcysteine Nicotinamide Glutamax
Require ECM (matrigel)	No	Yes	Yes
Constitute differentiated epithelium structure	Yes (ex vivo—already differentiated)	Yes (after 14–21 days culture)	Yes (duration to form not reported)
Orientation of apical ciliated side	Facing outwards (media)	Facing inwards (lumen)	Facing inwards (lumen)
Use of cultures	End-point experiments	End-point experiments	Can be passaged for on-going cultures (reported up to P18)
CFTR functional assay	Yes—spheroids shrink when CFTR is activated	Yes—spheroids swell when CFTR is activated	Yes—spheroids swell when CFTR is activated
Cryopreservation	No	No	Not reported
References	Bridges et al., 1991 Pedersen et al., 1999 Deslee et al., 2007 Guimbellot et al., 2017	Danahay et al., 2015 Hild and Jaffe, 2016 Brewington et al., 2018c	Sachs et al., 2018 Zhou et al., 2018

2D, Two-dimensional; ALI, Air-liquid interphase; BAL, Bronchoalveolar lavage; BEGM, Bronchial epithelial cell growth media; ECM, Extracellular matrix; FGF, Fibroblast growth factor; TGF-β, Transforming growth factor-beta.