Engineering Multilayered Hepatic Cell Sheet Model Using Oxygen-Supplying MeHA/CPO Hydrogel

Three-dimensional (3D) hepatic tissue engineering holds great potential for liver regeneration, disease modeling, and drug screening. These applications require densely layered hepatic tissues that mimic native 3D liver architecture. However, limited oxygen supply and reduced cell viability in densely layered hepatic constructs remain key challenges. To overcome this, this study developed a photo-crosslinkable, oxygen-releasing hydrogel composed of methacrylated hyaluronic acid (MeHA) and calcium peroxide (CPO). The MeHA/CPO hydrogel exhibited favorable rheological properties and sustained oxygen release. Induced pluripotent stem cell-derived hepatocyte (iHep) sheets were cultured with or without MeHA/CPO hydrogel in single- and double-layer formats. The hydrogel enhanced structural integrity and supported the formation of a multilayer (~33 µm). Double-layered iHep sheets with MeHA/CPO showed the significantly increased expression of paracrine factors (HGF, VEGF, Alb) and improved albumin secretion without loss of hepatocyte identity (AFP, HNF4α). This oxygen-releasing system effectively alleviates hypoxic stress, supporting the structural and functional viability of multilayered iHep sheets. Our platform provides a promising approach for engineering metabolically active hepatic tissues and may serve as a foundation for 3D hepatic tissue engineering.