Optimisation of culture conditions to develop an in vitro pulmonary permeability model.

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).