A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy

Abstract

Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy.

Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats.

Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins.

Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo. Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy.

Keywords: AEC, alveolar epithelial cell; AECI, type I alveolar epithelial cell; AECII, type II alveolar epithelial cell; AEpiCM, alveolar epithelial cell medium; AQP-5, aquaporin-5; Alveolar epithelial cell; Ca2+, ionized calcium; Cell sheet; FBS, fetal bovine serum; Feeder-free; GFP, green fluorescent protein; HBSS, Hanks' balanced salt solution; HE, hematoxylin and eosin; LCM, medium with a low ionized calcium concentration; PBS, phosphate-buffered saline; ROCK, rho kinase; Regenerative therapy; SP, surfactant protein; rLN511E8, recombinantly expressed laminin-511 E8 fragment.

Fig. 1

Schematic diagram of the methods used to fabricate and transplant alveolar epithelial cell (AEC) sheets. Lung tissues were carefully excised from male rats and finely minced, and cells were dissociated with elastase. A Percoll gradient was used to separate AECs from other cell types in the cell suspension. The isolated AECs were cultured under feeder-free conditions, and the expanded cells were seeded and cultured on temperature-responsive culture dishes. After subculture, the cells were harvested from the temperature-responsive dish as an AEC sheet by reducing the temperature from 37 °C to 20 °C for 30 min. Each AEC sheet was transplanted onto a partially resected region of the left lung or onto the left gluteal musculature of an athymic rat.

Fig. 2

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.

Fig. 3

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. 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.

Supplemental Fig. 1

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.