Primary type II pneumonocyte Isolation and culture

Pulmonary alveolar type II cells carry out highly specialized functions that include the synthesis, secretion, and reutilization of surfactant, a surface-active lipoprotein which acts to reduce surface tension at the alveolar air-liquid interface and is essential for normal breathing. Type II cells are unique in their ability to produce surfactant which contains relatively large amounts of dipalmitoylphosphatidylcholine (DPPC), a saturated glycerophospholipid with singular surface-active properties, and store surfactant lipids and proteins in organelles termed lamellar bodies.

1. Lung tissues were obtained.

2. The tissues were minced (1-2 mm³) in primary cell system culture.

3. After 5 days of organ culture with daily medium changes, lung explants were dissociated by digestion with primary cell isolation for 15 min at 37°C with vigorous pipeting.

4. Following digestion, the cells were treated with DEAE-dextran (250 µg/ml; Sigma Chemical Co.) and incubated for 45 min with shaking at 37°C.

5. DEAE-dextran treatment selectively eliminates fibroblasts.

6. The cells were pelleted at 400 × g and plated either onto T-25 flasks (5× 10⁵ cells per flask) coated with primary cell extracellular matrix (ECM).

7. The extracellular matrix-coated flasks were prepared from confluent monolayers of MDCK cells that were treated with deoxycholate (1%) for 5 min.

8. The flasks were washed three times with Hank's balanced salt solution and stored at 37°C until used.

9. Plated epithelial cell-enriched cultures were incubated overnight in primary cell medium with 10% fetal bovine serum.

10. Flasks were washed twice with medium to eliminate dead and non-adherent cells, and then incubated in primary cell medium without fetal bovine serum.

11. In studies where type II pneumonocytes were plated onto flasks, culture medium was placed either below or above and below the cell monolayer.

12. The plating density of the cells after overnight incubation was approximately 70-80%.

 






Reference

1. Alcorn JL, Smith ME, Smith JF, Margraf LR, Mendelson CR. Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses. Am J Respir Cell Mol Biol. 1997; 17:672-682.

2. Chinoy MR, Dodia C, Fisher AB. Increased surfactant internalization by rat type II cells cultured on microporous membranes. Am J Physiol Lung Cell Mol Physiol. 1993; 264: L300–L307.

3. Dobbs LG, Gonzalez R, Williams M. An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis. 1986; 134: 141–145.