Pulmonary type II cell lamellar body ultrastructure preserved by rapid freezing and freeze drying

Lamellar body ultrastructure was examined in cultured type II alveolar epithelial cells processed by a method of rapid freezing and freeze drying in the absence of both chemical fixation and solvent dehydration. This method of specimen preparation was chosen to optimize the retention of soluble substances within the type II cell. The use of cultured cell aggregates in which type II cells line the free surface facilitated the effectiveness of rapid freezing for the preservation of lamellar body fine structure. Lamellar bodies of frozen/frozen dried type II cells showed none of the often profound lipid extraction artifact produced by conventional processing. Instead they exhibited a substructure with noteworthy characteristics in common with lamellar bodies processed by resin dehydration lipid retention methods (Stratton, 1976). Importantly, the lamellae of frozen/frozen dried lamellar bodies were contiguous, with no interlamellar space, as is commonly observed in solvent-processed (extracted) specimens. The dimensions of lamellar components in frozen/frozen dried lamellar bodies were, however, different from published values for resin-dehydrated lipid-retained specimens. Lamellar width and the widths of component phospholipid head and fatty acid tail regions in frozen/frozen dried lamellar bodies were approximately 35% smaller than values reported for resin-dehydrated lamellar bodies. This difference was attributed to shrinkage of lamellar components as water was removed from the unfixed tissue during the freeze-drying process. Lamellar bodies preserved by rapid freezing/freeze drying to optimize the in situ retention of intracellular components possess closely adherent concentric membranous lamellae. This supports the contention that the widely appreciated lamellar pattern of the pulmonary lamellar body represents the in vivo molecular organization of intracellular surfactant phospholipids.