, the Netherlands). The remaining bioscaffold was processed for histology. Sections (20 μm thick) were stained for platelets with anti-integrin αIIb antibody (Santa

Cruz Biotechnology Inc., Santa Cruz, CA) and anti-mouse alexa fluor 633 secondary antibody. Cell nuclei were stained with YO-PRO1 (Invitrogen, Corp., Carlsbad, CA). The stained tissue was imaged with LSM510 confocal microscope (Carl Zeiss, Jena, Germany) and 4 pictures from each section BI 6727 manufacturer were taken. Average fluorescence from stained platelets was quantified in each picture with image analysis software SigmaScan Pro 5.0 (Aspire Software International, Ashburn, VA). Previous studies have shown that decellularization of thick tissues, such as liver, could be achieved only if the tissue was sectioned into thin slices and agitated in a detergent solution, which destroyed the organ’s architecture, including its vascular network.19 Perfusion of Triton X-100 and ammonium hydroxide containing solution successfully decellularized the livers of Ipatasertib various species including from mice (Fig. 1B), rats, ferrets (Fig. 1A), and adult pig.

After passing the decellularization solution through the vascular network (∼1 hour for mice, ∼2 hours for ferrets, ∼3 hours for rats, and ∼24 hours for pig livers), the liver parenchyma became transparent and the vascular tree was clearly visible under low magnification microscopy (Fig. 1B). Spectrophotometric analysis indicated the removal of approximately 97% of the DNA from the tissue, confirming the efficiency of the perfusion decellularization method (Supporting Information Fig. 1A). These results were further confirmed by agarose gel electrophoresis, followed by ethidium bromide

staining (Supporting Information Fig. 1B). To evaluate whether the ultrastructure of the bioscaffold was preserved after decellularization, we examined sections by scanning electron microscopy (SEM) (Fig. 1C-F). We observed reticular collagen fibers that provide support for the hepatic tissue (Fig. 1C). As shown in Fig. 1D, “portal triad” structures consisting of a large portal vein, bile duct, MCE公司 and the hepatic artery, remained intact. We observed honeycomb structures typical of the hepatic lobules surrounding the portal triad, but no remaining intact cells could be seen. Figure 1E shows multiple hollow structures at the liver hilum with intact and patent blood vessels; increased magnification revealed tightly interwoven fibers along the vessels (Fig. 1F). Histochemical staining was carried out to further characterize the composition of the decellularized tissue extracellular matrix (ECM). H&E staining showed pink eosinophilic staining typical of collagen, whereas no basophilic staining typical of cellular nuclear material was observed (Fig. 1G). Masson’s Trichrome staining confirmed these results, showing a homogeneous blue staining consistent with collagen (Fig. 1H).