Successful decellularization of thick-walled tissue: Highlighting pitfalls and the need for a multifactorial approach

Abstract

Decellularization of thick tissue is challenging and varying. Therefore, we tried to establish a multifactorial approach for reliable aortic wall decellularization. Porcine aortic walls were decellularized according to different procedures. Decellularization was performed for 24 (G1), 48 (G2), and 72 h (G3) with a solution of 0.5% desoxycholate and 0.5% dodecyl sulfate. The procedure was characterized using intermittent washing steps, the inclusion of sonication as well as DNase and α-galactosidase treatment. The decellularization efficiency was measured by the evaluation of 4',6-diamidino-2-phenylindole and hematoxylin and eosin staining and quantitative DNA assays. Pentachrome and picrosirius red staining, scanning electron microscopy as well as glycosaminoglycan assays were performed to evaluate the effect of the procedure on the extracellular matrix. 4',6-Diamidino-2-phenylindole and hematoxylin and eosin staining revealed a large amount of remaining nuclei in all groups. However, consecutive DNase treatment had a significant effect. While the remaining DNA was detected in some samples of G1 and G2, samples of G3 were fully decellularized. Glycosaminoglycan content was significantly reduced to 50% after 24 h (G1) but remained constant for G2 and G3. Picrosirius red staining revealed an intact and stable collagen network without any visible defects. Pentachrome staining substantiated these results. Nonetheless, the fiber network remains intact, which could be confirmed by reflection electron microscopy analysis. In this study, we developed a procedure that grants successful decellularization of porcine aortic wall while maintaining the fibrous microstructure. We highlighted the significant effect of DNase and α-galactosidase treatment. In addition, we could show the need for a multifactorial treatment and comprehensive evaluation protocols for thick tissue decellularization.