Isolation, biochemical characterization, and culture of lung type II cells of the rat

A method is described for the isolation of rat lung epithelial Type II cells using trypsin digestion of tissue to release cells for subsequent separation by Percoll gradient centrifugation. Both the concentration of trypsin and the age (body weight) of the rat affect the yield from primary digestion and the final number of Type II cells obtained. A lung weighing 1 g from a 200 g rat yields approximately 30 X 10(6) washed Type II cells (approximately 25% of the total estimated lung population). These cells have a plating efficiency of 40-50% after 48 h of culture. The cells have a high alkaline to acid phosphatase ratio (usually greater than 4.0) compared with that of alveolar macrophages (0.1) and accumulate putrescine by an active transport mechanism with an apparent KM between 8 and 14 microM. Together with studies of 3H]thymidine uptake into DNA, which is maximal between 48 and 72 h of culture, these quantitative measurements form a good basis for investigating the interactions between a number of chemical agents and Type II cells in vitro.