Abstract
Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.
Highlights
The extracellular matrix (ECM) plays an important role by regulating cell behavior through structural and biochemical stimulation
Even though trypsin + EDTA has been previously used in decellularization protocols, this method detached all tested samples and it is not suited for decellularization protocols on a glass slide
To quantify the different decellularization levels resulting from different treatments, pixels corresponding to tissue were separated from background pixels, quantified, and compared to native tissue pixel intensities as previously described (Narciso et al, 2021)
Summary
The extracellular matrix (ECM) plays an important role by regulating cell behavior through structural and biochemical stimulation. The interstitial ECM is in the intercellular spaces and is composed mostly of fibrous proteins and polysaccharides, most predominantly collagen type I and III, elastin, and fibronectin. The basement membrane is made up of sheets of deposition of ECM components—mainly collagen type IV and laminins—that are located under epithelial and endothelial cells (Pompili et al, 2021). These two layers make up the “core matrisome.”. It is unsurprising the growing interest to work on physiomimetic tissue scaffolds by decellularizing different types of tissues (Mendibil et al, 2020)
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