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

Area of Science:

• Biomaterials Science
• Regenerative Medicine
• Hepatology

Background:

• Three-dimensional (3D) hepatic tissue engineering is crucial for liver regeneration, disease modeling, and drug screening.
• Dense layering of hepatic tissues is required for native 3D liver architecture mimicry.
• Hypoxia and reduced cell viability in dense constructs are significant challenges.

Purpose of the Study:

• To develop a photo-crosslinkable, oxygen-releasing hydrogel to enhance 3D hepatic tissue engineering.
• To investigate the impact of the hydrogel on cell viability, structural integrity, and function of layered hepatic tissues.
• To overcome oxygen supply limitations in densely layered hepatic constructs.

Main Methods:

• Fabrication of a methacrylated hyaluronic acid (MeHA) and calcium peroxide (CPO) hydrogel.
• Characterization of hydrogel rheological properties and oxygen release kinetics.
• Culture of induced pluripotent stem cell-derived hepatocyte (iHep) sheets in single- and double-layer formats with and without the MeHA/CPO hydrogel.

Main Results:

• The MeHA/CPO hydrogel demonstrated sustained oxygen release and favorable rheological properties.
• The hydrogel enhanced structural integrity and supported the formation of multilayered iHep sheets (~33 µm).
• Double-layered iHep sheets with MeHA/CPO exhibited increased expression of paracrine factors (HGF, VEGF, Alb), improved albumin secretion, and maintained hepatocyte identity (AFP, HNF4α).

Conclusions:

• The oxygen-releasing hydrogel effectively alleviates hypoxic stress in multilayered hepatic tissues.
• This platform supports the structural and functional viability of engineered hepatic tissues.
• The developed hydrogel system offers a promising foundation for advanced 3D hepatic tissue engineering.