Liver diseases pose a major global health burden, underscoring the need for alternatives to conventional therapies and liver transplantation. Although hepatocyte transplantation holds therapeutic potential, its clinical application is limited by poor cryopreservation outcomes, including low post-thaw viability, freeze-thaw-induced damage, immune rejection, restricted cell viability, and low engraftment efficiency. In order to resolve these limitations, we propose a novel strategy for acute liver injury using polyphenol-engineered, lyophilized non-living hepatocytes that retain native morphology and preserve bioactive factors, inspired by the principle of dead cell therapy. Through a polyphenol-based process under high concentration and low temperature conditions, cellular viability is rapidly terminated while intracellular components are preserved. By applying this approach in combination with lyophilization, engineered hepatocytes function as both a biological reservoir and a transporter and consistently release hepatocyte-derived therapeutic factors such as hepatokines, which are important for liver regeneration.

 Primary hepatocytes were isolated from C57BL/6 mice using a two-step collagenase perfusion procedure. The isolated hepatocytes were engineered with polyphenol under defined conditions, frozen at -80 °C, and lyophilized for 24 hours using a lyophilizer. Interestingly, these engineered hepatocytes preserved total protein and RNA levels comparable to live cells and showed preserved morphology. In co-culture experiments, oxidatively stressed cells showed enhanced cell viability and reduced ROS levels when co-cultured with engineered hepatocytes. To evaluate the therapeutic effects of engineered hepatocytes in vivo, C57BL/6 mice were given an intraperitoneal injection of carbon tetrachloride (CCl₄) to cause acute liver injury. One million hepatocytes were transplanted through the hepatic portal vein 24 hours after CCl₄ was administered, and the mice were sacrificed 24 hours later for histological and biochemical analysis. Mice treated with engineered hepatocytes showed markedly improved histological recovery and improved liver function. In the CCl₄-treated group, cytoplasmic infiltration and necrosis were observed, but in the group transplanted with engineered hepatocytes, the structure was reconstructed without significant difference from the group transplanted with fresh hepatocytes.

 In conclusion, engineered non-living hepatocyte transplantation provides a stable and efficient alternative to existing hepatocyte transplantation in the treatment of acute liver injury. The results of this study show the potential as a treatment that can be used in the form of a ready-made product for various liver diseases.