Stable ciliary activity in human nasal epithelial cells grown in a perfusion system

Purpose: Explore the usefulness of a perfusion system in order to establish human nasal epithelial cell cultures suitable for long-term in vitro ciliary beat frequency (CBF) and cilio-toxicity studies. Methods: The cells were obtained by protease digestion of nasal biopsy material. The cells were plated at a density of 0.8–1 × 10 6 /cm 2 on Vitrogen-coated polyethylene terephthalate membranes, and cultured under submerged conditions in a CO2 incubator or in a perfusion system (initiated on days 8–9 after plating). The CBF was determined at 24.1 ± 0.8 ◦ Cb y a computerized microscope photometry system. The morphology of the cultured cells was characterized by transmission electron microscopy (TEM). Results: Under CO2 incubator culture conditions, stable ciliary activity was expressed and maintained from day 2 to day 24. Under perfusion system culture conditions, the CBF (mean ± S.D., n = 4) amounted to 8.4 ± 0.9 and 8.8 ± 0.4 Hz on days 7 and 14, respectively. These values were lower as compared to the corresponding CBF obtained in the CO2 incubator cultures (9.5 ± 0.6 and 9.9 ± 1.0 Hz, respectively). Reference cilio-stimulatory (glycocholate) and cilio-inhibitory (chlorocresol) compounds were used to assess CBF reactivity. In the CO2 incubator and 7- and 14-days perfusion system cultures, glycocholate (0.5%) showed a reversible cilio-stimulatory effect of 23, 26 and 21%, respectively, while chlorocresol (0.005%) exerted a reversible cilioinhibitory effect of 36, 40 and 36%, respectively. TEM revealed polarized cuboidal to columnar epithelial morphology, with well-differentiated ciliated cells under CO 2 and perfusion system conditions (up to day 23). Conclusion: Culturing human nasal epithelial cells on Vitrogen-coated polyethylene terephthalate membranes in submerged conditions in a CO2 incubator and in a perfusion system offers the possibility for long-term preservation (up to 22–24 days) of stable and reactive CBF in vitro. © 2004 Elsevier B.V. All rights reserved.