A Mn(2+)-enhanced, RGD-dependent adhesion technique for isolation of adult rat type II alveolar epithelial cells for immediate functional studies.

Abstract

This report describes a Mn(2+)-enhanced, RGD-dependent adhesion technique for isolation of adult rat type II cells for immediate functional studies. Lung cells were dissociated by 30 U/ml porcine pancreatic elastase and 50 micrograms/ml trypsin instilled in the airways. Macrophages were selectively removed by adhesion on purified normal goat IgG-coated petri dishes. Type II cells were isolated by adhesion for 45 min, on ProNectin-F-coated dishes in the presence of 0.5 mM Mn2+. The adherent type II cells were then detached with 0.025% trypsin, 2 mM EDTA in Hepes-buffered saline, pH 7.4. The technique yielded 1.5 to 1.7 x 10(7) (n = 8) cells from a 150- to 200-g rat. Greater than 90% of the cells were pure type II cells as judged by tannic acid staining and immunostaining with monoclonal antibody 4AmAb, which recognizes pneumocin, a type II cell marker. The technique reduced the time required for cell isolation from the current 16 to 24 h to 2 to 2.5 h, using commonly available laboratory equipment and reagents. Cells isolated by the procedure were used to study cell adhesion and spreading on purified extracellular matrix components in the presence of different divalent cations. Mn2+, Co2+, Ni2+, and Mg2+ enhanced adhesion of freshly isolated type II cells to fibronectin and ProNectin-F, while Ca2+ did not promote type II cell adhesion on these substrata. RGDS peptide at 1 mg/ml concentration inhibited the divalent cation-enhanced cell adhesion.