In vitro model to mimic the lung epithelial barrier for nano-toxicology studies.
Derk-R; Mishra-A; Stueckle-T; Friend-S; Castranova-V; Rojanasakul-Y; Chen-M; Wang-L
Toxicologist 2012 Mar; 126(Suppl 1):71
Previous data have shown that upon alveolar deposition, dispersed carbon nanotubes (CNT) can penetrate into the interstitium or pleural area which may indicate a specific mechanism of CNT induced pulmonary toxicity. Our hypothesis is that physicochemical properties of CNT could play a key role in determining the penetration mechanism of CNT into deep lung tissue. However, this would be difficult to determine in vivo. Lung epithelial cells typically form tight junctions which serve as a protective barrier against external particulates and can be monitored through resistance measurement in vitro. An experimental model was developed using an immortalized human lung epithelial cell line [Calu-3 cells (HTB- 55), ATCC, Manassas, VA] and Transwell ® inserts (Costar 6.5 mm polyester membrane with 3.0 um pores). Calu3 cells were cultured at 10, 20 or 50 x 103 per insert in Eagle's Minimum Essential Medium (EMEM) containing 15% Fetal Bovine Serum (FBS). After two days, medium was changed to EMEM that contained 2, 5, 10, or 15% FBS to assess serum effects on tight junction formation. Resistance (ohms) of the cell monolayer was monitored every day from days 3 - 18 in culture using the Epithelial Voltohmeter and STX2 electrode (World Precision Instruments). A subset of Transwell inserts were collected at various resistances and days in culture and tight junctions were immunofluorescence stained using the ZO-1 antibody. Tight junctions were observed using confocal microscopy after the resistance reached 2000 ohms in all concentrations of FBS tested. Resistance reached and maintained > 4000 ohms in 5 days at 50k, in 12 days at 20k and 14 days at 10k cell density. Establishment of Calu3 cell monolayers with functional tight junctions is dependent on filter size, seeding density and serum concentration. The Transwell Calu-3 model represents a potential rapid assessment in vitro model to test the effects of nano-material exposure on cell tight junction integrity.
Nanotechnology; Toxic-materials; Health-hazards; Immune-reaction; Immune-system; Cell-function; Pulmonary-clearance; Cellular-uptake; Toxic-effects; Respiratory-system-disorders; Pulmonary-system-disorders; Pleural-cavity; Lung-cells; Lung-disorders; Lung-function; Lung-tissue; Particulates; Serological-techniques; In-vitro-study; Immunological-tests; Filters; Exposure-assessment; Exposure-methods; Antibody-response; Analytical-models
The Toxicologist. Society of Toxicology 51st Annual Meeting and ToxExpo, March 11-15, 2012, San Francisco, California