Introduction
Hepatocyte sheet (HS) transplantation is expected to be an effective therapeutic strategy as an alternative to liver transplantation. We have developed a unique HS formation technique and have conducted study on the improvement of liver function (Miyamoto et al., Regen Ther. 2021). However, although HS has shown improvement in liver function for a short period of time, long-term functional improvement has not been achieved. This may be due to the lack of multidimensional structures in existing hepatocyte sheets, although the liver maintains various functions by possessing unique structures such as sinusoids and biliary ducts. In fact, the lack of biliary structures in the HS may induce liver damage due to bile stasis. (Fujii et al., J Tissue EngRegen Med, 2018). In other words, the formation of bile drainage mechanism is considered an important issue to maintain long-term liver function. In parallel, we have established a biliary drainage system with a luminal structure in vitro using chemically induced hepatic progenitor cells (CLiPs) (Huang et al., Biotechnol Bioeng. 2021).
Aims & Methods
In this study, we attempted to develop a new novel Hepato-Biliary Sheet (HBS) based on our biliary drainage system and evaluated its structure and hepatic function.Mouse embryonic fibroblasts (MEFs) and GFP-positive rat-derived CLiPs were sequentially seeded in temperature-responsive culture dishes and cultured for 2 weeks to induce the biliary drainage system. HBS was formed by seeding and culturing primary mature rat hepatocytes. The HBS was then implanted on the liver surface of a mouse model of liver failure, which was created by administration of Retrorsine and 70% hepatectomy, and its structure and function were evaluated in vivo.
Results
First, immunofluorescence image analysis of the prepared HBS revealed a structure consisting of CLiP-derived bile ducts (GFP-positive) and mature hepatocytes (Alb-positive, Glutamine-positive). To evaluate the incorporation of bile into the HBS, a fluorescent reagent (Cell Tracker Orange) as a marker substance was added to culture medium and bile excretion via hepatocytes bile duct cells was observed 24 hours later. Compared to HS prepared from mature rat hepatocytes alone, drug metabolism (CYP3A4 activity) and albumin production were significantly improved. CYP3A4 activities in HBS and HS were 139.3±59.9 and 75.4±47.1 (LUM/hr) (p<0.0001) and albumin productions were 1.5±0.5×104 and 1.1±0.1 x104 (ng /day) (p=0.005), respectively. Next, when the HBS was transplanted to the liver surface in non-liver failure mouse, it was confirmed that a bile duct-like structure (CK7-positive) was maintained and that mature hepatocytes (ALB-positive) were adjacent to the bile duct-like structure, and that vascular components (CD34-positive) derived from mice were infiltrated, confirming long-term cell viability, at least 1 month. Finally, to evaluate the effect of transplantation in a mouse model of liver failure, rat albumin concentration in blood was measured, and the HBS transplantation group showed higher albumin concentration than the HS group at 7 days (9.5×10^5±8.2×10^5 ng/mL vs 5.8×10^5±6.1×10^4ng/mL, P<0.05) and 28 days (1.1×10^6±9.2×10^5 ng/mL vs 3.2×10^5±3.2×10^5 ng/mL, P<0.05),respectively.
Conclusion
We have observed formations of hepatocyte and bile duct structure in HBS which is confirmed to have biliary drainage and drug metabolizing activities. HBS are expected to improve liver function, and to play a valuable role in the treatment of liver diseases through regenerative medicine.
References
Miyamoto D, Sakai Y, Huang Y, et al. Functional changes of cocultured hepatocyte sheets subjected to continuous liver regeneration stimulation in cDNA-uPA/SCID mouse: Differences in transplantation sites. Regen Ther. 2021;18:7-11.
Fujii M, Yamanouchi K, Sakai Y, et al. In vivo construction of liver tissue by implantation of a hepatic non-parenchymal/adipose-derived stem cell sheet. J Tissue Eng Regen Med. 2018;12:287-295.
Huang Y, Sakai Y, Hara T, et al. Bioengineering of a CLiP‐derived tubular biliary‐duct‐like structure for bile transport in vitro. Biotechnol Bioeng. 2021;118:2572-2584
Disclosure
This study was supported in part by the research grant from Eisai Co., Ltd.
