In-vitro nasal drug delivery studies: comparison of derivatised, fibrillar and polymerised collagen matrix-based human nasal primary culture systems for nasal drug delivery studies

Abstract

The aim of this study was to establish a collagen matrix-based nasal primary culture system for drug delivery studies. Nasal epithelial cells were cultured on derivatised (Cellagen membrane CD-24), polymerised (Vitrogen gel) and fibrillar (Vitrogen film) collagen substrata. Cell morphology was assessed by microscopy. The cells were further characterised by measurement of ciliary beat frequency (CBF), transepithelial resistance (TER), permeation of sodium fluorescein, mitochondrial dehydrogenase (MDH) activity and lactate dehydrogenase (LDH) release upon cell exposure to sodium tauro-24, 25 dihydrofusidate (STDHF). Among the three collagen substrata investigated, the best epithelial differentiated phenotype (monolayer with columnar/cuboidal morphology) occurred in cells grown on Cellagen membrane CD-24 between day 4 and day 11. Cell culture reproducibility was better with Cellagen membrane CD-24 (90%) in comparison with Vitrogen gel (70%) and Vitrogen film (< 10%). TER was higher in cells grown on Vitrogen gel than on Cellagen membrane CD-24 and Vitrogen film. The apparent permeability coefficient (Papp x 10(-7)cm s(-1)) of sodium fluorescein in these conditions was 0.45+/-0.08 (Vitrogen gel) and 1.91+/-0.00 (Cellagen membrane CD-24). Except for LDH release, CBF and cell viability were comparable for all the substrata. Based on MDH activity, LDH release, CBF, TER and permeation studies, Cellagen membrane CD-24- and Vitrogen gel-based cells were concluded to be functionally suitable for in-vitro nasal drug studies. Vitrogen film-based cultures may be limited to metabolism and cilio-toxicity studies.

Retinoic acid and hydrocortisone strengthen the barrier function of human RPMI 2650 cells, a model for nasal epithelial permeability

“…In our preliminary experiments several methods were tested, different peptides and also a collagen gel were used for surface coating. The best cell differentiation, functional polarisation, and transepithelial resistance were observed in the case of 0.05 v/v% rat tail collagen coated surfaces in agreement with previous findings (Agu et al 2001). This collagen matrix enhanced the attachment of RPMI 2650 cells and established proper surface for the nasal epithelial cell layers.…”

“…Serum may also contain factors that regulate the formation of tight junctions in epithelial cells (Hashimoto and Shimizu 1993). In agreement with these data, the use of serum at 5-10 % as a medium supplementation was recommended for better attachment and long-term viability in primary cultures of nasal epithelial cells (Usui et al 2000;Agu et al 2001). Longer incubation with serumsupplemented medium also induced squamous, terminal differentiation of human airway epithelial cells (Wu et al 1986).…”

“…The minimum required concentration of retinoic acid for airway cultures was suggested to be 0.3 lg/mL (Yoon et al 2000). Well-differentiated mucociliary phenotype of human nasal epithelium cultures was also reported without retinoids (Van Scott et al 1988;Werner and Kissel 1995;Agu et al 2001). Retinoic acid at 0.01 lg/mL concentration had no effect on a nasal model based on RPMI 2650 cells (Wengst and Reichel 2010).…”

“…The extracellular matrix is the physiological microenvironment for the epithelial cell migration, proliferation, and differentiation; it is an essential element in the development of a successful culture of human nasal epithelial cells (De Fraissinette et al 1995). The use of biological matrices, such as collagen, laminin, and matrigel is essential to obtain a stable, well-differentiated and reproducible nasal culture system (Agu et al 2001). In our preliminary experiments several methods were tested, different peptides and also a collagen gel were used for surface coating.…”

In vitro Nasal Cell Culture Systems for Drug Transport Studies

“…Various culture media and supplements are presented in Table II. For the human nasal epithelial cell culture, Ham's F-12 (Wu et al, 1985;Yankaskas et al, 1985), Bronchial epithelial growth medium (BEGM)/Dulbecco's modified Eagle's medium (DMEM) (Chen et al, 2001), BEGM (Koizumi et al, 2008), Ham's F12/DMEM (Agu et al, 2004;Mallant et al, 2009;Werner and Kissel, 1995), DMEM (Agu et al, 2001;Yoo et al, 2003) and BEGM/DMEM-F12 (Huh et al, 2010; have been used. Serum may also be added into the cell culture media, but it may induce variability in cell growth and squamous differentiation.…”