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Open Access Green as soon as Postprint is submitted to ZB.
Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue.
Am. J. Physiol. Lung Cell Mol. Physiol. 309, L323-L332 (2015)
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 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 4D multi-channel 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-labelled αSMA antibodies into the native cells of living 3D-LTCs, enabling live monitoring of αSMA-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 2D 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.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
4d Confocal Imaging ; Tissue-immunolabeling ; Precision Cut Tissue Slices; Cell-matrix Adhesions; In-vitro; Pulmonary-fibrosis; Migration; Slices; Culture; Compartments; Fibroblasts; Caveolin-1; Hydrogels
ISSN (print) / ISBN
1040-0605
e-ISSN
1522-1504
Quellenangaben
Volume: 309,
Issue: 4,
Pages: L323-L332
Publisher
American Physiological Society
Publishing Place
Bethesda, Md. [u.a.]
Non-patent literature
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Reviewing status
Peer reviewed