The conditionally reprogrammed cell technology for precision medicine in primary ciliary dyskinesia

Hintergrund Primary ciliary dyskinesia (PCD) is a congenital disease, leading to an impaiment of the mucociliary clearance. Culturing primary human epithelial cells (phNEC) at the air-liquid-interface (ALI) is the standard model for diagnostics and functional characterization. Challenges are a low yield and short lifespan of phNEC. A conditionally reprogrammed cell (CRC) system could overcome this, by enhancing cell growth and lifespan, while preserving the cell-of-origin function as previously shown for bronchial epithelial cells [1]. Thus, we aimed to establish and optimize the CRC technology for phNEC from people with PCD (pwPCD).

Methoden Nasal brushings were taken from 45pwPCD with genetically confirmed diagnosis. phNEC were cultured with irradiated ^3T3fibroblasts in the presence of Y-27632. After expansion, cells were partially cryopreserved (biobanked), further passaged and cultured at ALI for 21-35 days in PneumaCult™-ALI medium. Ciliary Beat Frequency (CBF) was assessed prior to culturing and at ALI. Mucociliary transport was assessed at ALI using fluorescent beads and time-lapse microscopy. Culture morphology and cell type composition were determined by immunofluorescence staining (IF).

Ergebnisse Utilizing the CRC method for patient-derived phNEC had an excellent success rate (> 90%) and yielded a high cell count (median: 10 x 10⁶ cells at passage 1, range: 2 x 10⁶to 27 x 10⁶ cells). Immunofluorescence staining revealed pseudostratified highly ciliated epithelial layers in differentiated ALI cultures. CBF showed comparable results in freshly obtained vs cultured nasal cells and mucociliary transport was significantly reduced in pwPCD at ALI.

Schlussfolgerung Culturing primary nasal epithelial cells from people with PCD utilizing the CRC technology stands out by its non-invasiveness, excellent success rate and high cell count yield, which allows successful biobanking. This method could be used for functional characterization as well as high throughput screening for therapeutic targets as a step towards personalized medicine for people with PCD.

Publication History

Article published online:
22 February 2024

• Literatur

• 1 Gentzsch M, Boyles SE, Cheluvaraju C, Chaudhry IG, Quinney NL, Cho C. et al. Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells. Am J Respir Cell Mol Biol 2017; 56 (05) 568-74