Abstract:

BACKGROUND: Type Ⅱ alveolar epithelial cells (AECⅡ) play an important role in lung development and regulation of lung function. It is closely related with the genesis and development of pulmonary fibrosis, lung cancer and so on. In order to make an in vitro study of these diseases, a stable and effective cell model for isolation, primary culture and identification of AECⅡ should be established.
OBJECTIVE: To develop a reliable method for the isolation, purification, primary culture and identification of AEC Ⅱ from mice, and provide a foundation for the further study of pulmonary fibrosis and lung cancer.
METHODS:  ①The lung tissue of mice was digested with low-concentration trypsin and collagenase Ⅰ. That is, lung tissues of mice were cut into small pieces, digested with trypsin, and terminated the digestion by adding DMEM containing fetal bovine serum. The cells were digested once again using trypsin, and adding collagenase Ⅰ after termination. ②AEC Ⅱ was purified by differential centrifugation and immune adherence for primary culture. The pneumocytes suspension was incubated in IgG culture plate, and liquid containing non-adherent cells were once again cultured in IgG culture plate, then supernatant were abandoned, and the cells were cultured in culture dish and culture plate. The morphology and growth characteristics of primarily cultured AEC Ⅱ were observed by an inverted microscope; the yield, purity and viability of AEC Ⅱ were determined. AEC Ⅱ was identified by immunocytochemical staining surfactant apoprotein A (SP-A) and SP-C, and the specific cell structure was observed under a transmission electron microscope.
RESULTS AND CONCLUSION: By this method, an amount of (4.8±1.2)×10⁶ AEC Ⅱ were harvested from each mouse with a purity of (85±2.4)% and a viability of (92±2.4)%. Under an inverted microscope, AEC Ⅱ in primary culture showed a shape of round or cube and an island-like growth. The immunocytochemistry showed that the cytoplasm presented buffy SP-A and green SP-C. Typical features of AEC Ⅱ including lamellar bodies and microvilli could be seen under a transmission electron microscope. The results demonstrated that, AECⅡ of great amount and high purity could be obtained by this method, which meets the requirement of a cell model establishing for further in vitro study.