Burgstaller Gerald, Vierkotten Sarah, Lindner Michael, Königshoff Melanie, Eickelberg Oliver
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
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.
Am J Physiol Lung Cell Mol Physiol. 2015 Aug 15;309(4):L323-32. doi: 10.1152/ajplung.00061.2015. Epub 2015 Jun 19.
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.
在过去几十年中，细胞行为的研究主要是在未包被或细胞外基质（ECM）包被的塑料培养皿中完成的。迄今为止，大量细胞生物学研究致力于在体外模拟体内天然微环境。对于肺而言，作为肺组织培养物（LTCs）进行离体培养的外植体提供了一种三维（3D）组织模型，其中所有细胞都处于其自然微环境中。然而，评估ECM与细胞组织成分之间动态实时相互作用的技术仍然缺乏。在此，我们描述了源自健康和纤维化小鼠肺以及患者来源的3D-LTCs的活3D-LTCs的特异性多维免疫标记，以及对其进行实时四维多通道成像。这种方法能够评估间充质细胞和巨噬细胞与其ECM之间的动态相互作用。此外，瞬时表达粘着斑标记物的成纤维细胞融入了3D-LTCs，为研究细胞粘着与其天然来源的ECM之间的动态相互作用开辟了新途径。一种新型蛋白质转移技术（FuseIt/Ibidi）将荧光标记的α-平滑肌肌动蛋白抗体转运到活3D-LTCs的天然细胞中，从而能够实时监测位于3D-LTCs纤维化病变中的天然组织肌成纤维细胞中α-平滑肌肌动蛋白阳性应力纤维。最后，该技术可应用于健康和患病的人肺组织，以及传统二维细胞培养中的贴壁细胞。这种新方法将为ECM（病理）生物学的动力学提供有价值的新见解，详细研究ECM与细胞组织成分在其自然微环境中的相互作用。
