Validation of a new live cell strain system: characterization of plasma membrane stress failure

Motivated by our interest in lung deformation injury, we report on the vali dation of a new live cell strain system. We showed that the system maintain s a cell culture environment equivalent to that provided by conventional in cubators and that its strain ouput was uniform and reproducible. With this system, we defined cell deformation dose (i.e., membrane strain amplitude)- cell injury response relationships in alveolar epithelial cultures and stud ied the effects of temperature on them. Deformation injury occurred in the form of reversible, nonlethal plasma membrane stress failure events and was quantified as the fraction of cells with uptake and retention of fluoresce in-labeled dextran (FITC-Dx). The undeformed control population showed virt ually no FITC-Dx uptake at any temperature, which was also true for cells s trained by 3%. However, when the membrane strain was increased to 18%, simi lar to5% of cells experienced deformation injury at a temperature of 37 deg reesC. Moreover, at that strain, a reduction in temperature to 4 degreesC r esulted in a threefold increase in the number of cells with plasma membrane breaks (from 4.8 to 15.9%; P < 0.05). Cooling of cells to 4<degrees>C also lowered the strain threshold at which deformation injury was first seen. T hat is, at a 9% substratum strain, cooling to 4 degreesC resulted in a 10-f old increase in the number of cells with FITC-Dx staining (0.7 vs. 7.5%, P < 0.05). At that temperature, A549 cells offered a 50% higher resistance to shape change (magnetic twisting cytometry measurements) than at 37<degrees >C. We conclude that the strain-injury threshold of A549 cells is reduced a t low temperatures, and we consider temperature effects on plasma-membrane fluidity, cytoskeletal stiffness, and lipid trafficking as responsible mech anisms.