Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia.
BACKGROUND
Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical surface facing the outside and accessible for analysis of ciliary function.


METHODS
We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls. Nasal ciliary cells and sheets were removed on day 1 by nasal brush biopsy and analyzed with regard to ciliary beat pattern-and frequency using high-speed video imaging for standard reference values. Three-dimensional explant spheroid formation was initiated in the same individual on the same day by incubation of cells and sheets from a separate brush biopsy. Harvested spheroids were analyzed earliest possible and values of spheroid ciliary beat pattern and frequency were compared to the corresponding reference values from day 1.


RESULTS
Spheroids formed fast in serum-free culture medium. Formation was successful in 15 out of 18 (82%) sampled individuals. Thus, formation was successful in seven healthy controls and eight PCD patients, while unsuccessful in 3 with PCD due to infection. Median (range) number of days in culture before harvesting of spheroids was 4 (1-5) in healthy versus 2 (1-5) in PCD. Spheroid ciliary beat pattern and frequency were unchanged compared to their corresponding day 1 standard reference values. Spheroid ciliary beat frequency discriminated highly significant between healthy controls (9.3 Hz) and PCD patients (2.4 Hz) (P< 0.0001). Survival of spheroids was 16 days in a single healthy person.


CONCLUSIONS
Patient-specific three-dimensional explant spheroid formation from a minimal invasive nasal brush biopsy is a feasible, fast and valid ex vivo method to assess ciliary function with potential of aiding the diagnosis of PCD. In addition, it may be a useful model in the investigation of pathophysiological aspects and drug effects in human nasal airway epithelium.
Danish PCD & chILD Centre, CF Centre Copenhagen, Paediatric Pulmonary Service, Department of Paediatrics and Adolescent Medicine, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, Copenhagen, DK-2100 Denmark.
June Kehlet
JK
Marthin
Danish PCD & chILD Centre, CF Centre Copenhagen, Paediatric Pulmonary Service, Department of Paediatrics and Adolescent Medicine, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, Copenhagen, DK-2100 Denmark.
Elizabeth Munkebjerg
EM
Stevens
Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
Lars Allan
LA
Larsen
Department of Biology, Section of Cell Biology and Physiology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark.
Søren Tvorup
ST
Christensen
Danish PCD & chILD Centre, CF Centre Copenhagen, Paediatric Pulmonary Service, Department of Paediatrics and Adolescent Medicine, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, Copenhagen, DK-2100 Denmark.
Kim Gjerum
KG
Nielsen
Airway epithelial; Cell culture; Cilia, ciliogenesis; Ciliary beat frequency; Ciliary beat pattern; Diagnosis; Primary ciliary dyskinesia; Three-dimensional explant spheroids