title: Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue.
abstract: During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment.
authors.affiliation: Comprehensive Pneumology Center, University Hospital of the Ludwig-Maximilians-University Munich and Helmholtz Zentrum München, Member of the German Center for Lung Research, Munich, Germany; and gerald.burgstaller@helmholtz-muenchen.de.
authors.forename: Gerald
authors.initials: G
authors.lastname: Burgstaller
authors.affiliation: Comprehensive Pneumology Center, University Hospital of the Ludwig-Maximilians-University Munich and Helmholtz Zentrum München, Member of the German Center for Lung Research, Munich, Germany; and.
authors.forename: Sarah
authors.initials: S
authors.lastname: Vierkotten
authors.affiliation: Center for Thoracic Surgery, Asklepios Biobank for Lung Diseases, Comprehensive Pneumology Center, Asklepios Clinic Munich-Gauting, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Lindner
authors.affiliation: Comprehensive Pneumology Center, University Hospital of the Ludwig-Maximilians-University Munich and Helmholtz Zentrum München, Member of the German Center for Lung Research, Munich, Germany; and.
authors.forename: Melanie
authors.initials: M
authors.lastname: Königshoff
authors.affiliation: Comprehensive Pneumology Center, University Hospital of the Ludwig-Maximilians-University Munich and Helmholtz Zentrum München, Member of the German Center for Lung Research, Munich, Germany; and.
authors.forename: Oliver
authors.initials: O
authors.lastname: Eickelberg
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D005260:Female; D006801:Humans; D021621:Imaging, Three-Dimensional; D008168:Lung / Q000378:metabolism / Q000473:pathology; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011658:Pulmonary Fibrosis / Q000378:metabolism / Q000473:pathology*; D013194:Staining and Labeling; D059008:Time-Lapse Imaging; D046509:Tissue Culture Techniques
chemical_list: D000199:Actins
keywords: 4D confocal imaging; three-dimensional ex vivo lung tissue cultures; tissue-immunolabeling