ABSTRACT
An in vitro model to study pulmonary translocation was created, using the human cell line Calu-3 and primary rat type II pneumocytes. Cells were seeded on permeable membranes with a 0.4 microm or 3 microm pore size, utilizing different culture conditions such as medium formulation and cell density. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. When seeded on inserts with 0.4 microm pore size, the Calu-3 cells and primary rat type II pneumocytes created high TEER values of 949+/-182 Omega cm(2) and 400+/-257 Omega cm(2), respectively. On membranes with 3 microm pores, Calu-3 cells achieved a high TEER value of 500+/-95 Omega cm(2). Our experiments indicate that the culture medium was more critical than the cell density, regarding the influence on TEER values. For both cell types a reduction of serum in the medium resulted in a decrease in TEER value. We established a good ('tight') monolayer of primary type II pneumocytes in Waymouth medium at a cell density of 0.9x10(6) cells/cm(2); the Calu-3 cells should be grown in DMEM medium containing Hepes at 0.75x10(6) cells/cm(2).
Subject(s)
Cell Culture Techniques , Cell Membrane Permeability , Respiratory Mucosa/metabolism , Administration, Inhalation , Animals , Biological Transport , Bronchi/metabolism , Cell Line , Cell Membrane/metabolism , Electric Impedance , Epithelial Cells/metabolism , Fluorescein/pharmacokinetics , Humans , Lung/metabolism , Permeability , Rats , Respiratory Mucosa/cytologyABSTRACT
Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007+/-300 and 348+/-62 Omega cm2 were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 microm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 microm pores, a TEER value of 241+/-90 Omega cm2 was reached on day 5; on 3 microm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 microm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 microm pores no translocation was observed, on 3 microm pores approximately 6% translocation was observed both for carboxyl- and amine-modified particles.
