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Equine airway smooth muscle culture: a model for the study of pathogenesis of recurrent airway obstruction (RAO)
Author(s) -
Steib Crystal,
Truax Robert,
Lomax Larry,
Venugopal Changaram
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a407-b
Subject(s) - desmin , staining , immunohistochemistry , pathology , airway , actin , cell culture , anatomy , biology , microbiology and biotechnology , medicine , vimentin , surgery , genetics
Recurrent airway obstruction (RAO) is a debilitating disease that affects horses. The objective of this project was to generate a protocol for the growth and maintenance of equine airway smooth muscle (E‐ASM) cells in culture to facilitate the study of airway diseases. The E‐ASM cells were collected aseptically, grown in culture media, and frozen at 6th passage. Cells were thawed and transferred to fresh medium, incubated at 37°C and 5% CO 2 and passaged every 3–4 days. On passages 10, 12, and 15 approximately 100,000 E‐ASM cells were transferred into a four‐chambered slide. These slides were incubated 5 days allowing a monolayer cells to attach to the slide. Cell confirmations were carried out using H&E staining for morphology and testing for actin and desmin using immunohistochemical (IHC) staining. The 10th passage cells were stained with H & E and the 12th and 15th passage were stained for smooth muscle actin and desmin. H&E staining confirmed the airway smooth muscle morphology. The 12th passage cells showed approximately 20% staining for actin, while no cells stained positive for desmin. However, at the 15th passage, the actin (approximately 50%) and desmin stained. We believe cells did not express desmin in the early passages due to the point of differentiation of the cells in culture. The study demonstrates that the protocol used in this project is ideal for culturing E‐ASM cells. Funded by NIH‐5 T35 RR017504 ‐03 & EHSP.