4, Stanford, Stanford, California, United States
3, Stanford, Stanford, California, United States
5, Stanford, Stanford, California, United States
2, Nanyang Technological University, Singapore, , Singapore
Hydrogels are useful biomaterials whose mechanical and transport properties may be tuned via choice of polymer and cross-link density. We have utilized an inverse colloidal crystalline array of sacrificial polystyrene spheres of 140µm diameter followed by photopolymerization of a hydrogel network in the interstices of the poragen array to generate a three-dimensional tissue scaffold. Choice of poragen diameter and annealing conditions allows control of the scaffold cavity size as well as the open channels connecting adjacent cavities. When poly(ethylene glycol)-diacrylate is used as the macromonomer, it is necessary to functionalize the surface of the resulting PEG scaffold because of the well-known resistance of PEG chains to protein and cell adhesion. Through chemical coupling of collagen I we were able to create microenvironments in which human umbilical vein endothelial cells as well as freshly isolated fetal total liver cells demonstrated good attachment and growth. We have demonstrated the formation of a 3D interconnected architecture, sustaining liver-specific functions (e.g., albumin secretions, cytochrome P450 activity and viral infectivity) over a very long term. Recently, we have advanced this system to fabricate very thin hydrogel layers that allow better oxygen and nutrient diffusion while maintaining the 3D interconnected structure. This has allowed us to culture human adult primary hepatocytes, the gold standard for liver studies. These cells are highly oxygen-sensitive and have intrinsically weak cell-cell and cell-extracellular matrix interactions. The new scaffold resulted in a dramatic change in cell morphology and function, increasing the prospect of constructing highly functional adult hepatocyte-based 3D liver tissue. In addition, we have used biodegradable materials, e.g., GelMA or fibrin, in place of PEG for a transplantation study on mice to show proof-of-concept for clinical use. A highly organized biodegradable scaffold was successfully developed even with the very soft hydrogel. We are currently using 3D printing of encapsulated endothelial cells to generate vascularized liver tissue. This approach eventually may lead to a readily transplantable organ or a long-term sustainable authentic in vitro human liver model that could be used for drug screening and disease modeling.