Rapid isolation of type II pneumocytes with magnetic removal of macrophages.
A method is described for the rapid preparation of lung cell fractions enriched in type II alveolar pneumocytes. Isolated perfused rabbit lungs are exposed to Fe3O4 by tracheal lavage, which permits pulmonary alveolar macrophages to phagocytize the particles. Alveolar epithelial cells are then selectively freed from the basement membrane matrix by critical placement of collagenase and elastase. Detached cells are harvested either by repeated tracheal lavage or by mincing the lobes and filtering freed cells through a series of nylon mesh sieves. Iron oxide-containing macrophages are then removed from the harvested cells by a strong magnetic field. A final sizing of the macrophage-depleted suspension yields a preparation enriched in alveolar type II cells. Eight million viable cells (95% type II) were obtained per rabbit lung when harvested by lavage, while 32 x 10(6) (88% type II) cells were obtained from minced lungs. These values for cell yield and relative purity are comparable to previously described separation methods that depend upon differences in cell density or size. A major advantage of the magnetic separation procedure is the substantially shortened preparation time, typically 2 hr instead of 4. The viability (90-95%), oxygen consumption (88 nmol/10(6) cells/hr), and incorporation of [14C]acetate and [14C]choline (0.44 and 0.115 nmol/10(6) cells/hr, respectively) indicate that these cells will be suitable for pharmacologic and toxicologic investigation.
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002498:Centrifugation; D052203:Ferrosoferric Oxide; D007501:Iron; D050356:Lipid Metabolism; D008264:Macrophages / Q000166:cytology* / Q000502:physiology; D008280:Magnetics; D008297:Male; D010087:Oxides; D010101:Oxygen Consumption; D010587:Phagocytosis; D011650:Pulmonary Alveoli / Q000166:cytology*; D011817:Rabbits
D010087:Oxides; D007501:Iron; D052203:Ferrosoferric Oxide