Isolation of human primary airway smooth muscle
Isolation of human primary airway smooth muscle
1. Primary cultures of human primary airway smooth muscle (ASM) cells were prepared from explants of airway smooth muscle.
2. The human trachea tissue was transported to the laboratory in primary cell culture medium containing 10% fetal calf serum (FCS), primary cell culture supplements, penicillin G (100 u ml−1), streptomycin (100 μg ml−1), amphotericin B (2.5 μg ml−1) and L-glutamine (4 mm).
3. The human trachea tissue was then washed 5 times in the same medium.
4. The trachea muscle was then dissected free of epithelium and connective tissue under sterile conditions.
5. Small (2 × 2 mm) explants of airway smooth muscle were then excised and about 10 explants placed in small dishes.
6. After explants were allowed to adhere, primary cell culture medium, containing FCS, antibiotics, primary cell culture supplements, amphotericin B and L-glutamine, was added to cover the explants.
7. The explants were incubated in humidified 5% CO2/95% air at 37°C.
8. The medium was changed every 3 days.
9. Smooth muscle cells were usually seen about 7 days later.
10. When cells were about to become confluent in some parts of the dish, the explants were removed.
11. Once confluent, cells were trypsinized with 0.25% trypsin and 0.02% EDTA in phosphate-buffered saline (PBS), centrifuged and resuspended in the above medium, counted and plated out in several 75 cm2 flasks and grown to confluence.
12. Cells were then detached with trypsin-EDTA, resuspended in 90% FCS + 10% dimethyl sulphoxide at a density of 106 cells ml−1, frozen in liquid nitrogen and stored until required.
13. Cells were thawed before use and plated at a density of 2 × 105 cells/well in 25-flask culture plates containing the above medium.
References
1. Hall, I.P., Widdops, S., Townsend, P. & Daykin, K. (1992). Control of cyclic AMP levels in primary culture of human tracheal smooth muscle cells. Br. J. Pharmacol., 107, 422–428.
2. Widdop, S., Daykin, K. & Hall, I.P. (1993). Expression of muscarinic M2 receptors in cultured human airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol., 9, 541–546.
3. Green, S.A., Turki, J., Bejarano, P., Hall, I.P. & Liggett, S.B. (1995). Influence of β2-adrenergic receptor genotypes on signal transduction in human airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol., 13, 25–33.
