Electron microscopic analysis of the mouse and human alveolar epithelial glycocalyx using high-pressure freezing and freeze substitution.

Alveolar epithelial cells (AECs) are covered apically by the alveolar lining layer (ALL). The alveolar epithelial glycocalyx (AEGCX), which is part of the ALL, consists of a mixture of glycoproteins, proteoglycans, and glycosaminoglycans. In comparison with the endothelial glycocalyx (GCX), much less is known about the composition and structure of the AEGCX, although it is presumed to be involved in many cellular processes. The challenges of in vivo visualization of the AEGCX, coupled with the distorting effects of aldehyde fixation, make it difficult to accurately determine its structural organization and molecular composition. In this study, we aimed to improve the preservation of the ultrastructure of the AEGCX by using high-pressure freezing combined with freeze substitution (HPF/FS) for transmission electron microscopy (TEM) analysis. HPF is a type of cryopreservation that uses both liquid nitrogen and high pressure. By using HPF/FS to preserve commercially available human primary AECs, human alveolar-like organoids, monolayers of human organoid-derived alveolar-like epithelial cells, and ex vivo tissue from both mice and human lungs, we observed two distinct regions on the apical surface via TEM: an inner GCX-like layer along the cell membrane followed by a loosely arranged outer layer. Our data show a bilayered structure on the surface of AECs. To minimize freeze-induced damage during HPF, a protein-rich cryoprotectant was introduced to stabilize the sample. By processing control samples of protein-rich cryoprotectants lacking cells, we found that HPF/FS preserves components of the cryoprotectant, as confirmed by TEM analysis. This implies that HPF/FS preserves not only cellular-derived constituents but also components found in the cryoprotectant, which may interfere with interpretation of the outer surface layer.NEW & NOTEWORTHY The structure of the AEGCX is not fully understood. To avoid formaldehyde-induced disruption of the AEGCX, we preserved samples by HPF/FS for TEM analysis. We uncovered two morphologically distinct regions on the apical surface of AECs. An inner GCX-like layer along the membrane followed by an associated outer layer. We found that cryoprotectants used during HPF to avoid freezing induced damage interfere with the outer layer, potentially misleading interpretations of its structure.