Quantifying Proliferation of Cultured Human and Rabbit Airway Smooth Muscle Cells in Response to Serum and Platelet-derived Growth Factor

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Abstract
Development of suitable methods for the quantification of the proliferative response of airway smooth muscle (ASM) cells in culture will assist the investigation of the cellular mechanisms underlying the hyperplasia and hypertrophy of ASM as seen in asthmatic airways. In this study, two rapid and simple colorimetric assays have been modified to enable the growth of human bronchial and rabbit tracheal smooth muscle in culture to be assessed. One method depends upon the reduction by living cells of the tetrazolium salt MTT to form a blue formazan product, whereas the other relies on rapid binding of the dye Coomassie brilliant blue to protein at acidic pH. Experiments demonstrated the validity of both assays in quantifying the proliferative response of cultured human and rabbit ASM cells. The increase in optical density observed for each assay correlated directly, throughout the duration of culture, with the increase in cell number determined by hemocytometry in human and rabbit ASM cells proliferating in response to fetal calf serum (1.25 to 10%). This relationship held also for rabbit tracheal ASM cells proliferating in response to the heterodimer of platelet-derived growth factor (1 to 50 ng/ml). Application of these methods to adherent proliferating cultures of human and rabbit ASM cells demonstrated their adaptability to the generation of growth curves in response to serum and to a defined growth factor. These methods allow both total cellular protein and proliferation to be estimated in human and rabbit ASM cells in culture, using assays that are rapid, reproducible, inexpensive, and easy to perform while negating the use of radioisotopes. It is intended that these additional methods should be useful in delineating some of the mechanisms that might contribute to the proliferative response of these cells--particularly since there has been a resurgence in interest in culturing smooth muscle cells derived from the airways.