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Journal ArticleDOI

An improved method for isolating type II cells in high yield and purity.

14 May 2015-The American review of respiratory disease (American Lung Association)-Vol. 134, Iss: 1, pp 141-145
TL;DR: Type II cells isolated by "panning" adhered more rapidly and completely in tissue culture than did cells isolate by centrifugation over discontinuous density gradients of metrizamide.
Abstract: A method has been developed for isolating alveolar type II cells by digesting lung tissue with elastase and “panning” the resultant cell suspension on plates coated with IgG. This method provides both high yield and purity of type II cells. In 50 experiments with rats, we obtained 35 ± 11 × 106cells/rat, 89 ± 4% of which were type II cells (mean ± SD). Type II cells isolated by “panning” adhered more rapidly and completely in tissue culture than did cells isolated by centrifugation over discontinuous density gradients of metrizamide. The “panning” method is superior to other methods for isolating type II cells in that it provides a population of type II cells of both high yield and high purity. The method is fast, reproducible, and easily adaptable to isolating type II cells from species other than rats.
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Journal ArticleDOI
30 Nov 1990-Science
TL;DR: Mechanical factors can trigger complex cellular events in nonneuron, nonmuscle cells and may be involved in regulating normal lung functions by stimulating type II cells to secrete surfactant.
Abstract: Deep inflation of the lung stimulates surfactant secretion by unknown mechanisms. The hypothesis that mechanical distension directly stimulates type II cells to secrete surfactant was tested by stretching type II cells cultured on silastic membranes. The intracellular Ca2+ concentration was measured in single cells, before and after stretching. A single stretch of alveolar type II cells caused a transient (less than 60 seconds) increase in cytosolic Ca2+ followed by a sustained (15 to 30 minutes) stimulation of surfactant secretion. Both Ca2+ mobilization and exocytosis exhibited dose-dependence to the magnitude of the stretch-stimulus. Thus, mechanical factors can trigger complex cellular events in nonneuron, nonmuscle cells and may be involved in regulating normal lung functions.

488 citations

Journal ArticleDOI
TL;DR: It is shown that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI), and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.
Abstract: We have shown that the integrin alphavbeta6 activates latent TGF-beta in the lungs and skin. We show here that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI). Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. TGF-beta directly increased alveolar epithelial permeability in vitro by a mechanism that involved depletion of intracellular glutathione. These data suggest that integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ALI and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.

459 citations

Journal ArticleDOI
TL;DR: This review addresses many of the issues involved in isolating and culturing type II cells, including the choice of a method of isolating cells, the importance of using specific markers of the differentiated type II cell phenotype, and the problems of maintaining these differentiated phenotypic characteristics in tissue culture.
Abstract: The alveolar type II cell performs many important functions within the lung, including regulation of surfactant metabolism, ion transport, and alveolar repair. Because type II cells comprise only 15% of all lung cells, it is difficult to attribute specific functions to type II cells from studies of whole lungs or mixed cell cultures. At the present time, there is no passaged line that exhibits the full range of known type II cell functions. For these reasons, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology. This review addresses many of the issues involved in isolating and culturing type II cells, including the choice of a method of isolating cells, the importance of using specific markers of the differentiated type II cell phenotype, and the problems of maintaining these differentiated phenotypic characteristics in tissue culture.

391 citations

Journal ArticleDOI
TL;DR: An in vitro system in which changes in alveolar epithelial cell viability can be measured after exposure to tightly controlled and physiologically relevant deformations is developed, and the relative effect of deformation frequency, duration, and amplitude on cell viability is reported.
Abstract: The onset of ventilator-induced lung injury (VILI) is linked to a number of possible mechanisms. To isolate the possible role of alveolar epithelial deformation in the development of VILI, we have developed an in vitro system in which changes in alveolar epithelial cell viability can be measured after exposure to tightly controlled and physiologically relevant deformations. We report here a study of the relative effect of deformation frequency, duration, and amplitude on cell viability. We exposed rat primary alveolar epithelial cells to a variety of biaxial stretch protocols, and assessed deformation-induced cell injury quantitatively, using a fluorescent cell viability assay. Deformation-induced injury was found to depend on repetitive stretching, with cyclic deformations significantly more damaging than tonically held deformations. In cyclically deformed cells, injury occurred rapidly, with the majority of cell death occurring during the first 5 min of deformation. Deformation-induced injury was increased with the frequency of sustained cyclic deformations, but was not dependent on the deformation rate during a single stretch. Reducing the amplitude of cell deformations by superimposing small cyclic deformations on a tonic deformation significantly reduced cell death as compared with large-amplitude deformations with the same peak deformation.

335 citations


Cites methods from "An improved method for isolating ty..."

  • ...The lungs were then excised, and ATII cells were dissociated and isolated in a technique adapted from Dobbs and coworkers (19)....

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  • ...After 1 h of incubation, the type ATII cells were isolated from the macrophages and contaminating cells by “panning” (19)....

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Patent
05 Nov 1992
TL;DR: In this paper, a method for transforming pulmonary cells as a means for treating disorders of the lung as well as for delivering substances systematically following expression in the lung has been proposed, but the method is not described.
Abstract: Methods and compositions for producing a mammal capable of expressing an exogenously supplied gene in cells of the airway are disclaimed. Liposome-nucleic acid complexes are prepared then delivered via aerosol to the lung airway. The invention provides a direct method for transforming pulmonary cells as a means for treating disorders of the lung as for providing a means for delivering substances systematically following expression in the lung.

323 citations