In vivo generation of thick, vascularized hepatic tissue from collagen hydrogel-based hepatic units

In vivo engineering of hepatic tissue based on primary hepatocytes offers new perspectives for the treatment of liver diseases. However, generation of thick, three-dimensional liver tissue has been limited by the lack of vasculature in the tissue-engineered constructs. Here, we used collagen hydrogel as a matrix to generate engineered hepatic units to reconstitute three-dimensional, vascularized hepatic tissue in vivo. Hepatocytes harvested from Sprague-Dawley rats were mixed with liquid type I collagen, concentrated Dulbecco's modified Eagle's medium (2 x), and hepatocyte maintenance medium to create hepatocyte/collagen hydrogel constructs. The constructs were then dissociated into cylindrical hepatic units (diameter/height: 2000-4000 microm/500-1000 microm). Stacking of hepatic units under the subcutaneous space resulted in significant cell engraftment, with the formation of large fused hepatic system (more than 0.5 cm thickness) containing blood vessels. In contrast, only less cell engraftment could be achieved when hepatocytes were transplanted in a manner of whole constructs. Functional maintenance of the engineered hepatic tissue was confirmed by the expression of liver-specific mRNA and proteins. The engineered hepatic tissue has the ability to respond to the regenerative stimulus. In conclusion, large hepatic tissue containing blood vessels could be engineered in vivo by merging small hepatic units. This approach for tissue engineering is simple and represents an efficient way to engineer hepatic tissue in vivo.