A method for culturing canine tracheal smooth muscle cells in vitro: Morphologic and pharmacologic observations

Abstract

A method of culturing canine tracheal smooth muscle cells in vitro is described. The morphology of these cells is monitored up to 60 days in culture and selected stages are illustrated. The characteristics of these cells are numerous mechanical attachments, the presence of thick filaments in suitably processed cells, and their contractile response to in vitro administration of carbachol, a cholinomimetic drug. They also possess nexus formations and both thin (actin) filaments and 10-nm filaments. Mitosis is found in the nonconfluent preparations up to 16 days after culturing. Cultures of 2 to 8 days appear to be most useful as pharmacological test vehicles. This system will be used to explore the phenomenon of adrenergic beta-2 receptor desensitization in airway smooth muscle, to attempt to localize these receptor sites and to determine how receptor affinity and/or number may be regulating cell response to pharmacologic agents.

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma

“…The ability of isolated or cultured smooth muscle cells to contract or relax in response to specific agonists is well documented [30,31] and airway smooth muscle cells are no exception. AVNER et al [32] reported that subconfluent primary cultures of canine tracheal smooth muscle cells contracted in response to the cholinomimetic, carbachol. Maintenance of this contractile phenotype in cultured smooth muscle cells is dependent upon a number of carefully controlled cell culture conditions, specifically cell density, avoidance of foetal bovine serum exposure and a requirement for heparin [14,33].…”

Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma

“…The techniques available for quantifying this muscle thickening can loosely be divided into in situ studies, involving morphometric analysis of the bronchial tree from normal and asthmatic patients [3][4][5][6][7], and studies employing cell culture techniques [17][18][19][20] as an in vitro model of the smooth muscle present in the intact airway wall.…”

“…This is compounded by modulations in phenotype, which may result in the loss of critical features of the differentiated cell. However, with the increasing realization of the role of smooth muscle in airway wall thickening and subsequent development of bronchial hyperresponsiveness, various groups have succeeded in culturing airway smooth muscle cells from a number of species, such as canine [17,18,27,28], bovine [29][30][31], rabbit [32][33][34], guinea-pig [35,36], sheep [37], and human [19,20,[38][39][40][41][42]. Although many of these initial concerns have been largely overcome, it remains essential to characterize fully the properties of these cells and to make comparisons where possible with the intact tissue.…”

“…Enzymatic dispersion is the technique adopted by most investigators to obtain primary cultures of airway smooth muscle cells from a carefully dissected preparation of large or small airway [17,19]. This method is used to obtain isolated smooth muscle cells in a morphologically well-differentiated and contractile state [23,24], where the immediate cellular environment may be rigorously controlled, especially when determining the effects of soluble or insoluble factors on cellular phenotype.…”

“…Contamination of primary cultures of airway smooth muscle cells by nonmuscle cell types can be more troublesome and should always be borne in mind when interpreting data. The most frequently reported cell contaminants are airway epithelial cells, microvascular and lymphatic endothelial cells and fibroblasts [17,18]. Epithelial and endothelial cells, both cobblestone in appearance, tend not to survive after a few days in culture, and are invariably lost following passaging from primary culture ( [18], and Hirst, unpublished observations).…”