Evaluation of human nasal RPMI 2650 cells grown at an air-liquid interface as a model for nasal drug transport studies.
This study tests the hypothesis that human nasal RPMI 2650 cells grown at an air-liquid interface is a feasible model for drug transport studies via the nasal route. RPMI 2650 cells were cultured in Eagle's minimal essential medium (MEM) at both air-liquid and liquid-liquid interfaces. For each culture regimen, monolayer integrity was tested by measuring the transepithelial resistance (TEER) as well as the transport of paracellular and transcellular markers across the monolayer. The expression of tight junction proteins-differentiation markers-in cells of the different monolayers was studied by western blot analysis and confocal microscopy. The highest TEER values (192 +/- 3 Omega . cm2) were observed for RPMI 2650 cells seeded onto collagen-coated permeable polytetrafluoroethylene inserts and grown at an air-liquid interface for 10 days; a seeding density of 4 x 10(5)/cm2 generated and maintained a cell monolayer with suitable barrier properties at days 9-12. Microscopic examination showed that RPMI 2650 cells grown on filter inserts formed a fully confluent monolayer. The apparent permeability coefficients of the paracellular marker, [14C] mannitol, and the transcellular marker, [3H] propranolol, were 5.07 +/- 0.01 x 10(-6) cm/s and 16.1 +/- 0.1 x 10(-6) cm/s, respectively. Western blot analysis indicated the presence of four tight junction proteins: ZO-1, occludin, claudin-1 and E-cadherin; and the quantities of ZO-1, occludin, and E-cadherin were significantly higher in cells grown at an air-liquid interface than in cells grown at a liquid-liquid interface. Confocal microscopic studies showed ZO-1, F-actin, occludin and claudin-1 proteins at cell-cell contacts and revealed significant differences in the distributions and densities of ZO-1 protein in cells grown at the two types of interface. The data indicate that RPMI 2650 cells grown at an air-liquid interface form polarized monolayers with the cells interconnected by tight junction proteins. This human nasal cell line model could provide a useful tool for in vitro screening of nasal drug candidates.
Bai
Shuhua
S
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 Coulter Drive, Amarillo, TX 79106, USA.
Yang
Tianzhi
T
Abbruscato
Thomas J
TJ
Ahsan
Fakhrul
F
D000388:Air; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009296:Nasal Cavity / Q000166:cytology / Q000378:metabolism*; D010599:Pharmacokinetics