Fig 2: Fabrication of alveolar epithelial cell (AEC) sheets. (a) AECs were subcultured on 35-mm-diameter temperature-responsive culture dishes in alveolar epithelial cell medium (AEpiCM) containing a rho kinase (ROCK) inhibitor (10 mM Y-27632). However, after 7 days of subculture, confluent AECs could not be harvested as a cell sheet when the temperature was reduced from 37 °C to 20 °C for 30 min. Immunohistochemical analysis revealed that cells cultured under these conditions did not express E-cadherin. (b) AECs were subcultured on 35-mm-diameter temperature-responsive culture dishes in AEpiCM containing a ROCK inhibitor, and the cells were confirmed to be confluent after 7 days. Then, the AECs were cultured for a further 3 days in AEpiCM containing a ROCK inhibitor and supplemented with 200 mg/L calcium chloride. Under these conditions, the AECs could be harvested as a cell sheet by reducing the temperature from 37 °C to 20 °C for 30 min. Immunohistochemical analysis revealed that cells cultured using this protocol expressed E-cadherin. (c–h) Hematoxylin and eosin (HE) staining and immunofluorescence staining of AEC sheets fabricated under the feeder-free conditions described in (b). Positive staining for aquaporin-5 (AQP-5), surfactant protein (SP)-A, SP-C, SP-D and Axin-2 showed that the cultured cells were AECs. Cross-sectional (upper panel) and planar (lower panel) views were obtained for the immunohistochemical analyses.

Fig 3: Immunohistochemical analyses of lung tissues without transplanted green fluorescent protein (GFP)-expressing alveolar epithelial cell (AEC) sheets. (a) No staining was observed when the primary antibodies were omitted in negative control experiments. (b–e) Immunofluorescence staining of lung tissues for aquaporin-5 (AQP-5), surfactant protein (SP)-A, SP-C, SP-D and Axin-2.

Fig 4: Analyses of green fluorescent protein (GFP)-expressing alveolar epithelial cell (AEC) sheets (GFP-AEC sheets) after transplantation onto the lung. (a) Three GFP-AEC sheets were transplanted onto a partially resected area of the left lung of an athymic rat. (b) Seven days later, illumination of the transplantation region with fluorescent light revealed the presence of GFP-positive cells, which were derived from the transplanted cell sheets. (c–i) Histological and immunohistochemical analyses of lung tissues and transplanted GFP-AEC sheets. The lung tissues and transplanted GFP-AEC sheets were subjected to HE staining or immunofluorescence staining for GFP, aquaporin-5 (AQP-5), surfactant protein (SP)-A, SP-C, SP-D or Axin-2. Staining with HE demonstrated that the transplanted AEC sheets were tightly adhered to the lung tissue, and no air spaces were found between the cell sheets and the pulmonary parenchyma (arrowheads). The immunohistochemical analyses revealed that cells on the lung surface expressed GFP (white arrows), and the same region also expressed AQP-5, SP-A, SP-C, SP-D and Axin-2. No staining was observed when the primary antibodies were omitted in negative control experiments.

Fig 5: Alveolar epithelial cells (AECs) cultured under feeder-free conditions. (a) Primary AECs were cultured under feeder-free conditions in the absence or presence of a rho kinase (ROCK) inhibitor (10 mM Y-27632) and in the absence or presence of recombinantly expressed laminin-511 E8 fragment (rLN511E8) as a coating on the tissue culture dish (i.e., four experimental groups). The cultured AECs were harvested 7 days later, and the number of cells was counted. (b) Comparison of the number of AECs expanded under each feeder-free condition. The data are displayed as box plots showing the median, interquartile range and range (n = 7). *p < 0.05. (c) Phase-contrast microscopy of AECs cultured on an rLN511E8-coated dish in alveolar epithelial cell medium (AEpiCM) containing a ROCK inhibitor. The cultured AECs exhibited a polygonal, cobblestone-like morphology characteristic of epithelial cells. (d–h) Immunohistochemical analyses of AECs cultured on rLN511E8-coated dishes in AEpiCM containing a ROCK inhibitor. The cultured AECs were immunostained using primary antibodies against aquaporin-5 (AQP-5), surfactant protein (SP)-A, SP-C, SP-D and Axin-2.