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

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 μm or 3 μm 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 μm pore size, the Calu-3 cells and primary rat type II pneumocytes created high TEER values of 949 ± 182 Ω cm 2 and 400 ± 257 Ω cm 2, respectively. On membranes with 3 μm pores, Calu-3 cells achieved a high TEER value of 500 ± 95 Ω 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 0.9 × 10 6 cells/cm 2; the Calu-3 cells should be grown in DMEM medium containing Hepes at 0.75 × 10 6 cells/cm 2.