A major roadblock to successful organ bioengineering is the need for a functional vascular network within the engineered tissue. Here, we describe the fabrication of three‐dimensional, naturally derived scaffolds with an intact vascular tree. Livers from different species were perfused with detergent to selectively remove the cellular components of the tissue while preserving the extracellular matrix components and the intact vascular network. The decellularized vascular network was able to withstand fluid flow that entered through a central inlet vessel, branched into an extensive capillary bed, and coalesced into a single outlet vessel. The vascular network was used to reseed the scaffolds with human fetal liver and endothelial cells. These cells engrafted in their putative native locations within the decellularized organ and displayed typical endothelial, hepatic, and biliary epithelial markers, thus creating a liver‐like tissue in vitro. Conclusion: These results represent a significant advancement in the bioengineering of whole organs. This technology may provide the necessary tools to produce the first fully functional bioengineered livers for organ transplantation and drug discovery. (HEPATOLOGY 2011;53:604‐617)

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Background and Aims The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5)–positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Approach and Results Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40‐fold cell expansion after 2 weeks, compared with 6‐fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver‐like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up‐regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. Conclusions We established a highly efficient method for culturing large numbers of LGR5‐positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

The use of synthetic and naturally-derived scaffolds for bioengineering of solid organs has been limited due to a lack of an integrated vascular network. Here, we describe fabrication of a bioscaffold system with intact vascular tree. Animal-donor organs and tissues, ranging in size up-to thirty centimeters, were perfused with decellularization solution to selectively remove the cellular component of the tissue and leave an intact extracellular matrix and vascular network. The vascular tree demonstrated sequential fluid flow through a central inlet vessel that branched into an extensive capillary bed and coalesced back into a single outlet vessel. In one example, the liver, we used central inlet vessels to perfuse human and animal liver cells through the bioscaffold to create a functional liver tissue construct in vitro. These results demonstrate a novel yet simple and scalable method to obtain whole organ vascularized bioscaffolds with potential for liver, kidney, pancreas, intestine and other organs' bioengineering. These bioscaffolds can further provide a tool to study cells in their natural three-dimensional environment, which is superior for drug discovery platform compared with cells cultured in two-dimensional petri dishes.