A method for isolating adult and neonatal airway smooth muscle cells and measuring shortening velocity

Abstract

Methods are described for isolating smooth muscle cells from the tracheae of adult and neonatal sheep and measuring the single-cell shortening velocity. Isolated cells were elongated, Ca2+ tolerant, and contracted rapidly and substantially when exposed to cholinergic agonists, KCl, serotonin, or caffeine. Adult cells were longer and wider than preterm cells. Mean cell length in 1.6 mM CaCl2 was 194 ± 57 (SD) μm ( n = 66) for adult cells and 93 ± 32 μm ( n = 20) for preterm cells ( P < 0.05). Mean cell width at the widest point of the adult cells was 8.2 ± 1.8 μm ( n = 66) and 5.2 ± 1.5 μm ( n = 20) for preterm cells ( P < 0.05). Cells were loaded into a perfusion dish maintained at 35°C and exposed to agonists, and contractions were videotaped. Cell lengths were measured from 30 video frames and plotted as a function of time. Nonlinear fitting of cell length to an exponential model gave shortening velocities faster than most of those reported for airway smooth muscle tissues. For a sample of 10 adult and 10 preterm cells stimulated with 100 μM carbachol, mean (± SD) shortening velocity of the preterm cells was not different from that of the adult cells (0.64 ± 0.30 vs. 0.54 ± 0.27 s−1, respectively), but preterm cells shortened more than adult cells (68 ± 12 vs. 55 ± 11% of starting length, respectively; P < 0.05). The preparative and analytic methods described here are widely applicable to other smooth muscles and will allow contraction to be studied quantitatively at the single-cell level.