1A). The choices of the rinse media or the buffers for the nucleases can be any of a number of options as long as the salt concentration and ionic strength are such as to maintain the collagens and associated matrix components in an insoluble state. The choice of the delipidation method is also critical to be effective and yet should be gentle. We chose a combination of sodium deoxycholate (SDC) and phospholipase A2 (PLA2) to rapidly degrade the phosphoglyceride check details located on the cytoplasm membrane and mitochondrial membrane into lysolecithin, a powerful surfactant, which can induce necrosis and cytolysis. The reactive

formula is shown in the Supporting Fig. S1. We avoided prolonged exposure of the scaffolds to the enzymes from the disrupted cells during delipidation and the high salt washes because they can greatly decrease the content of elastin and the content of glycosaminoglycans (GAGs) such as heparan sulfates (HS), Protease Inhibitor Library cell assay chondroitin sulfates (CS), dermatan sulfates (DS), and heparins (HP), sites at which cytokines

and growth factors bind.29 We used soybean trypsin inhibitor and careful control of the pH (7.5-8.0) and time (30-60 minutes) to limit the activity of the proteases derived from disrupted cells. We perfused the whole tissue through relevant vasculature (e.g., portal vein in the liver), enabling us to rapidly isolate (within a few hours) a biomatrix scaffold with minimal loss of matrix components.

The rapidity of the IMP dehydrogenase isolation is due to the initial step with detergent that delipidates the tissue within ≈30-60 minutes (not hours or days as in protocols used by others, see Supporting Table 5). The resulting biomatrix scaffolds are translucent or white (Fig. 1D). Moreover, using this perfusion method we maintained the primary vasculature channels, portal and hepatic vein, and most of the vascular branches in the liver, which increased the decellularization efficiency (Fig. 1E). Fluorescent rhodamine-labeled dextran particles perfused through the biomatrix scaffolds remained within the remnants of the vasculature, demonstrating that they are patent (Fig. 1E1). There is a progressive flow of the dye from large vessels to the fine blood vessel branches along the channels without leakage (demonstrated even more dramatically in the Supporting Video). This fact will be helpful in the future in revascularization of scaffolds as a means of preparing engineered tissues for either three-dimensional culture and/or for implantation ex vivo. When sectioned, scaffolds retain the histological structure of the original tissue, including the recognizable remnants of major histological entities such as blood vessels, bile ducts, and Glisson’s capsule (GC). Compare Fig.