Background : To comprehend the pathophysiology of liver diseases, it is important to implement regeneration processes in vivo. In previously reported studies, mouse chemically induced hepatic progenitors (mCdHs) demonstrated liver regeneration in the disease mouse model. It has not been revealed whether there is any difference between the single-cell mixture of mCdHs and the three-dimensional (3D) structured microtissues with endothelial cells. This study aims to apply a 3D bioprinting approach to the liver disease model, to confirm transplant feasibility, and validate its effects.

Methods : To generate mCdHs from adult Td-Tomato EGFP mice, primary mouse hepatocytes (mPHs) were isolated and cultured in reprogramming media for 7 days. The mCdHs were characterized in comparison to mPHs, and their long-term maintenance and hepatic differentiation potential were successfully confirmed in vitro. We fabricated three types of microtissue spheroids from mCdHs and mouse endothelial cells (mECs) using bioprinting technology and confirmed liver regeneration after transplanting them in vivo.

Results : Single-cell mixtures of mCdHs and vascularized microtissue spheroids (core-shell, segmented, non-structured) were transplanted into the livers of FAH-/- and FRG-/- mice. Biochemical analysis results on day 10 showed that AST, ALT, and T.BIL levels decreased in all groups compared to the PBS group (negative control). Notably, the single-cell group showed relatively higher AST and ALT levels. Immunohistochemistry and H&E staining revealed positive expression rate differences in the transplanted liver area.

Conclusions : This study utilizing 3D bioprinting and mCdHs demonstrated promising results in terms of hepatocyte function and supported the potential of utilizing liver disease modeling and transplantation therapies.