The tissue culture/biophysical Core in this program project serves three key roles in this PPG and is a key mechanism for integrated collaboration between the projects. First, the Core will provide a centralized resource facility for eight lung endothelial cell cultures used in each of five projects. Specifically, the cell culture core will continue provide freshly isolated and cultured murine lung endothelial EC, cell lines of human and bovine pulmonary artery EC, human and bovine pulmonary microvascular EC, human dermal microvascular EC, native cell lines and cell lines over-expressing wild type or tyrosine-deficient mutant of protein tyrosine phosphatase mu, and rat fat pad microvascular cells over-expressing protein kinase C-delta. Second, this Core will utilize sophisticated state of the art technologies to carefully expose lung pulmonary endothelium in mechanically active settings relevant to physiological and pathophysiological in vivo conditions. Important tools for this work include culturing EC under conditions of controlled cyclic stretch and controlled laminar shear stress. Finally, the Core will quantitate the physiological significance of each of the studies proposed in the Program Project: (1) measurements of albumin clearance across confluent EC, (2) measurements of trans-monolayer electrical resistance of static cultures and cyclic stretch-preconditioned EC monolayers using endothelial cell-substrate impedance sensing system (ECIS); (3) real time simultaneous measurements of EC trans-endothelial electrical resistance under shear stress; and (4) EC wound assay with real time monitoring of EC wound healing using ECIS lectroporation/wounding module. The extensive experience of the biophysical and tissue culture core personnel continue to function as a tissue culture core (previous Program Project Grant HL-58064) assuring the continuous supply of high quality cells, culturing cells under precisely characterized conditions of shear stress or cyclic stretch and expert assistance in quantitation of EC monolayer barrier function. The close proximity of the tissue culture and biophysical facilities will provide reproducibility and consistency of results obtained in the Program Projects, and ensure the most economical use of the Core to adequately supply all the investigators of the Program Project.
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