Isolation of bovine type II pneumocytes in high yield and purity

A method has been developed for the isolation of bovine type II pneumocytes by enzymatic tissue dissociation and subsequent density gradient centrifugation. After mechanical defibrination, the crude cell suspension contains 71.9 +/- 26.6 X 10(7) cells (viability greater than 95%) with a type II cell purity of 39.4 +/- 10.9%. Due to their low buoyant density, bovine type II pneumocytes can be purified in a single step to 95.7 +/- 1.7% by discontinuous density gradient centrifugation on a 1.040 g/ml Percoll gradient. Isolated cells are identified by light and fluorescence microscopy that show their characteristic intracytoplasmatic surfactant granules. The fine structure of the surfactant lamellar bodies is examined in ultra thin sectioned and freeze-fractured type II pneumocytes. Isolation of bovine type II pneumocytes for the in vitro study of the surfactant system offers an alternative to the use of laboratory animals and provides an ideal system for the isolation of any desired cell number from one animal by simply increasing the size of the lung segment to be trypsinized.