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Alveolar epithelial cells type II (Imperial College School of Science)

Human alveolar epithelial cells type II are characterized by a cuboidal appearance, apical microvilli, lamellar bodies and well-formed tight junctions between all the cells.

Alveolar type II cells can be isolated from normal regions of lung tissue obtained following lobectomy in lung carcinoma patients. The tissue is washed with sterile NaCl (0.15M), cut into 5cm3 pieces and placed in a Petri dish. Trypsin solution (10–15 mL/5cm3 piece) is instilled into the lung tissue for 15 min at 370C.  Trypsinization is repeated 2 more times for a total duration of 45 min. The tissue is finely minced into 1–2mm3 pieces in the presence of NBCS (or FSC; 10 mL/5 cm3 piece), added to inhibit trypsin. DNase I (250-μg/mL HBSS; 7 mL/ 5cm3 piece) is added to the suspension and shaken vigorously for 5 min. The tissue suspension is then filtered through a large gauge mesh (400–500μm) and then through a 40-μm mesh to remove undigested tissue and debris from the enzymatically-released epithelial cells, which pass through the filter. Filtrate is centrifugated at 300g for 10 min at 120C. The cell pellet is resuspended in 30mL LPHM/HBSS (50%/50%) containing DNase I (100 mg/mL) and plated into either T-75 or T-175 culture flasks incubated for 1–2 h at 370C. The medium containing nonadherent, type II cell-enriched cell population is removed and centrifuged at 300g for 10 min at 120C. The cell pellet is resuspended in 30mL complete  NBCS (10%) culture medium and incubated in a T-75 tissue culture flask for 1–2 h at 370C for contaminating fibroblasts adherence. The nonadherent, type II cell-enriched medium are removed and centrifuged. Cell pellets from each piece of tissue are resuspended in complete medium at a density of 1×106 epithelial cells/mL, plated onto Vitrogen-coated plates and incubated in 5% CO2 at 370C for 24 h. After 24 h, the medium and nonadherent cells are removed. Fresh complete medium is then applied to the remaining loosely attached cells. After another 16–24 h, the medium is removed, remaining loose cells are washed off with HBSS and fresh complete medium is applied. The cells form a confluent monolayer within 3 days of plating.

See additional Stem, Progenitor & Primary Cells for: Epithelial Cells , Lung
Primary Cell
Homo sapiens
Alveolar epithelial cells type II