3D analysis of cellular interactions with extracellular matrix in living lung tissue
The researchers used Imaris to create this Surface rendering of lung fibroblasts fully repopulating an emphysematous decellularized mouse lung slice. After 5 days the cells were fixed and stained for phalloidin. Courtesy of Gerald Burgstaller, Helmholtz Zentrum München, Gerald Burgstaller and colleagues, Comprehensive Pneumology Center (CPC), Helmholtz Zentrum München
The extracellular matrix (ECM) provides structure and support to cells in tissues and organs and is known to play a key role in cell development, tissue repair, and disease. However, scientists don’t fully understand the nature of this interplay. Researchers led by Gerald Burgstaller, Helmholtz Zentrum München are using decellularized lung tissue scaffolds and Imaris software to learn more about how the ECM affects cells in various microenvironments in the lung.
To study how the ECM interacts with cells, the researchers created a unique 3D ECM model by decellularizing excised mouse lung tissue scaffolds from diseased and healthy mouse lungs. They then reseeded the scaffolds with fibroblast cells, the main producers of the ECM. Using Imaris, the researchers observed and analyzed the reseeded cells in various ECM microenvironments, including those in alveolar regions, airways, vasculature, and the mesothelium as well as in emphysematous or fibrotic ECM.
“Imaris is our daily-routine workhorse,” said Burgstaller. “We use it for depicting large 4D datasets, displaying Surface rendered datasets of lung tissue and cells, quantifying cell migration and cell invasion with the Spots module, and measuring morphological features of single cells.”
Getting a 3D view of repopulation
This Surface rendering shows the morphology of one cell that repopulated the alveolar niche. The cell was transfected with an empty EGFP vector. The dataset represents a confocal z-stack. Courtesy of Gerald Burgstaller, Helmholtz Zentrum München.
By using Imaris to create Surface renderings from 3D confocal z-stacks, the researchers showed that the decellularized lung scaffolds are repopulated by fibroblasts not only on the surface but also within the inner core of the scaffolds. They also observed fibroblasts “homing” to the interstitial ECM. Fibroblasts are usually found in the lung’s interstitial space, located between the epithelial lining and the endothelial cells of the vasculature.
The researchers used the Imaris Spots function to quantify cell migration, showing that different ECM niches within the lung tissue have different effects on cell migration. This analysis also revealed that the migration of reseeded fibroblasts was significantly slowed in fibrotic and alveolar microenvironments.
“We found that that the inherent physical or compositional characteristics of different microenvironments, or niches, of the ECM within the lung distinctly influences the behaviour of reseeded cells,” said Burgstaller. “We also compared the native ECM microenvironments with fibroblasts cultured on 2D plastic dishes and observed striking differences in protein expression, cellular morphologies, and migration.”
Obtaining more realistic data
This Surface rendering shows a different angle for a cell that repopulated the alveolar niche. Courtesy of Gerald Burgstaller, Helmholtz Zentrum München.
The new ECM models developed by the researchers could be useful for a variety of applications. For example, they might improve drug discovery and mechanistic studies because using cells cultured in ECM more closely mimic in vivo or in situ environments and might, therefore, provide more relevant hits or reveal different pathways than studies using cells cultured on 2D plastic dishes. Understanding how cells behave in decellularized lung scaffolds might also reveal whether decellularizing lungs and then repopulating them with stem cells might offer a feasible method to engineer transplantable lungs.
As a next step, the researchers are using Imaris to aid in their work to figure out the mechanisms involved in the cellular behaviour differences seen for various niches of the lung’s ECM. They plan to attempt isolating certain ECM niches to create a distinct system for studying cell behaviour.
Research paper: Burgstaller G, Sengupta A, Vierkotten S, Preissler G, Lindner M, Behr J, Königshoff M, Eickelberg O. 2018. Distinct niches within the extracellular matrix dictate fibroblast function in (cell-free) 3D lung tissue cultures. Am J Physiol Lung Cell Mol Physiol. doi: 10.1152/ajplung.00408.2017