Isolation, culture et caractérisation phénotypique de cellules musculaires lisses endobronchiques équines
Endobronchial smooth muscle is one of the main stakeholder in asthma by alteration of his volume and phenotype. Sampling airway smooth muscle using endobronchial biopsies is performed in order to study pathways associated with remodeling, but currently, little is known regarding the stability of the ASM phenotypes during in vitro culture. Specifically, whether the contractile phenotype is maintained in culture and his evolution across passages is not established. Two problems could explain this lack of information: first the sampling, secondly the maintenance of contractile phenotype in culture.     We developed culture condition based on tracheal smooth muscle in the aim to ensure the upkeep of contractile phenotype. Then we developed a sampling protocol to harvest and maintain in culture ASM cells from endobronchial biopsies on a live animals. The stability of the ASM contractile phenotype was evaluated at each passage using immunofluorescence, FACS and western blot for α-sm-actin, desmin and myosin contents over a 2-month period. No significant differences were observed between the first and the fourth last passage (4th to 7th). 96.4% of the tracheal smooth muscle cell expressed α-sm-actin, 83.3 and 77% of cells expressed desmin and myosin respectively, at the 7th passage. Endobronchial smooth muscle expressed 84% of α-sm-actin, 57% of desmin and 69% of myosin, respectively.   Contractile phenotype was maintained and we determined evolution of contractile phenotype in standardize conditions that will provide a characterized support for coming research.
Biopsie Airway smooth muscle Biopsy Contractile phenotype Characterization Horses