Hepatocyte transplantation is expected to be an alternative therapy to liver transplantation; however, poor engraftment is a severe obstacle to be overcome. The adipose tissue-derived stem cells (ADSCs) are known to improve engraftment of transplanted pancreatic islets, which have many similarities to the hepatocytes. Therefore, we examined the effects and underlying mechanisms of ADSC cotransplantation on hepatocyte engraftment.

Because of the fragility of isolated hepatocytes, extremely poor engraftment of transplanted hepatocytes remains a severe issue in hepatocyte transplantation. Therefore, improving hepatocyte engraftment is necessary to establish hepatocyte transplantation as a standard therapy. Since the pancreatic islets are known to have favorable autocrine effects, we hypothesized that the transplanted islets might influence not only the islets but also the nearby hepatocytes, subsequently promoting engraftment. We evaluated the effects of islet co-transplantation using an analbuminemic rat model (in vivo model). Furthermore, we established a mimicking in vitro model to investigate the underlying mechanisms. In an in vivo model, the hepatocyte engraftment was significantly improved only when the islets were co-transplanted to the nearby hepatocytes (p < 0.001). Moreover, the transplanted hepatocytes appeared to penetrate the renal parenchyma together with the co-transplanted islets. In an in vitro model, the viability of cultured hepatocytes was also improved by coculture with pancreatic islets. Of particular interest, the coculture supernatant alone could also exert beneficial effects comparable to islet coculture. Although insulin, VEGF, and GLP-1 were selected as candidate crucial factors using the Bio-Plex system, beneficial effects were partially counteracted by anti-insulin receptor antibodies. In conclusion, this study demonstrated that islet co-transplantation improves hepatocyte engraftment, most likely due to continuously secreted crucial factors, such as insulin, in combination with providing favorable circumstances for hepatocyte engraftment. Further refinements of this approach, especially regarding substitutes for islets, could be a promising strategy for improving the outcomes of hepatocyte transplantation.

No optimal methods for short-term hepatocyte preservation have been established. We have recently developed a prominent oxygen-permeable bag (Tohoku Device [TD]) for pancreatic islet culture and transplantation. In this study, we investigated whether TD is also effective for hepatocyte preservation and tried to optimize other conditions.

Clinical hepatocyte transplantation (HTx) is only performed without general anesthesia, while inhalation anesthetics are usually used in animal experiments. We hypothesized that isoflurane may be a possible reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx. Syngeneic rat hepatocytes (1.0 × 107) were transplanted to analbuminemic rats with (ISO group) and without (AW group) isoflurane. The serum albumin, AST, ALT, LDH levels and several inflammatory mediators were analyzed. Immunohistochemical staining and ex vivo imaging were also performed. The serum albumin levels of the ISO group were significantly higher in comparison to the AW group (p < 0.05). The serum AST, ALT, LDH levels of the ISO group were significantly suppressed in comparison to the AW group (p < 0.0001, respectively). The serum IL-1β, IL-10, IL-18, MCP-1, RNTES, Fractalkine and LIX levels were significantly suppressed in the ISO group. The ischemic regions of the recipient livers in the ISO group tended to be smaller than the AW group; however, the distribution of transplanted hepatocytes in the liver parenchyma was comparable between the two groups. Isoflurane may at least in part be a reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx.

No optimal assay for assessing isolated hepatocytes before hepatocyte transplantation (HTx) has been established, therefore reliable and rapid assays are warranted. Isolated rat hepatocytes were dipped in a water bath (necrosis model), and were also cultured with Okadaic acid (apoptosis model) or vehicle, followed by cellular assessment including trypan blue exclusion (TBE) viability, ADP /ATP ratio, plating efficiency (PE), DNA quantity and ammonia elimination. Hepatocytes were transplanted into the liver of analbuminemic rats, subsequently engraftment was assessed by serum albumin and the histology of transplanted grafts. In the necrosis model, the ADP/ATP ratio was strongly and negatively correlated with the TBE (R2 = 0.559, P < 0.001). In the apoptosis model, the ADP/ATP ratio assay, PE, DNA quantification and an ammonia elimination test clearly distinguished the groups (P < 0.001, respectively). The ADP/ATP ratio, PE and DNA quantity were well-correlated and the ammonia elimination was slightly correlated with the transplant outcome. TBE could not distinguish the groups and was not correlated with the outcome. The ADP/ATP ratio assay predicted the transplant outcome. PE and DNA quantification may improve the accuracy of the retrospective (evaluations require several days) quality assessment of hepatocytes. The ADP/ATP ratio assay, alone or with a short-term metabolic assay could improve the efficiency of HTx.

Intraportal injection is regarded as the current standard procedure of hepatocyte transplantation (HTx). In islet transplantation, which shares many aspects with HTx, recent studies have clarified that instant blood-mediated inflammatory reaction (IBMIR), characterized by strong innate immune responses, can cause poor engraftment, so other transplant sites to avoid such a reaction have been established. Although IBMIR was reported to occur in HTx, few reports have evaluated alternative transplant sites for HTx. In this study, we sought to determine the optimum transplant site for HTx. Rat hepatocytes (1.0 × 107) were transplanted at the 9 transplant sites (intraportal (IPO), intrasplenic (IS), liver parenchyma, subcutaneous, intraperitoneal, renal subcapsular, muscle, inguinal subcutaneous white adipose tissue, and omentum) of analbuminemic rats. The serum albumin levels, immunohistochemical staining (albumin, TUNEL, and BrdU), and in vivo imaging of the grafts were evaluated. The serum albumin levels of the IPO group were significantly higher than those of the other groups (p < .0001). The BrdU-positive hepatocyte ratio of liver in the IS group (0.9% ± 0.2%) was comparable to that of the IPO group (0.9% ± 0.3%) and tended to be higher than that of the spleen in the IS group (0.5% ± 0.1%, p = .16). Considering the in vivo imaging evaluation and the influence of splenectomy, the graft function in the IS group may be almost entirely achieved by hepatocytes that have migrated to the liver. The present study clearly showed that the intraportal injection procedure is more efficient than other procedures for performing HTx.