The safety and short-term efficacy of hepatocyte transplantation (HCTx) have been widely proven. However, issues such as reduced viability and/or function of hepatocytes, insufficient engraftment, and lack of a long-term effect have to be overcome for widespread application of HCTx. In this study, we evaluated hepatocyte spheroids (HSs), formed by self-aggregation of hepatocytes, as an alternative to hepatocytes in single-cell suspension. Hepatocytes were isolated from C57BL/6 J mice liver using a three-step collagenase perfusion technique and HSs were formed by the hanging drop method. After the spheroids formation, the HSs showed significantly higher mRNA expression of albumin, ornithine transcarbamylase, glucose-6-phosphate, alpha-1-antitrypsin, low density lipoprotein receptor, coagulation factors, and apolipoprotein E (ApoE) than 2 dimensional (2D)-cultured hepatocytes ( p < 0.05). Albumin production by HSs was significantly higher than that by 2D-cultured hepatocytes (9.5 ± 2.5 vs 3.5 ± 1.8 μg/dL, p < 0.05). The HSs, but not single hepatocytes, maintained viability and albumin mRNA expression in suspension (92.0 ± 2.8% and 1.03 ± 0.09 at 6 h). HSs (3.6 × 106 cells) or isolated hepatocytes (fSH, 3.6 × 106 cells) were transplanted into the liver of ApoE knockout (KO-/-) mice via the portal vein. Following transplantation, serum ApoE concentration (ng/mL) of HS-transplanted mice (1w: 63.1 ± 56.7, 4w: 17.0 ± 10.9) was higher than that of fSH-transplanted mice (1 w: 33.4 ± 13.0, 4w: 13.7 ± 9.6). In both groups, the mRNA levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, MCP-1, and MIP-1β) were upregulated in the liver following transplantation; however, no significant differences were observed. Pathologically, transplanted HSs were observed as flat cell clusters in contact with the portal vein wall on day 7. Additionally, ApoE positive cells were observed in the liver parenchyma distant from the portal vein on day 28. Our results indicate that HS is a promising alternative to single hepatocytes and can be applied for HCTx.

Introduction The roles of preformed anti‐HLA donor‐specific antibodies (DSAs) in liver transplantation remain controversial. We evaluated the impact of preformed DSAs in living donor liver transplantation. Methods Adults who underwent living donor liver transplantation (n = 175) in our institute were included in this study. Lymphocyte cytotoxicity test (LCT), flow cytometric crossmatch (FCXM), and single‐antigen bead assays were performed. Results Among adult living donor liver transplantation recipients, 27 (16.5%) and 14 (8.5%) had pretransplant FCXM‐positive findings and LCT‐positive findings, respectively. FCXM‐positive patients displayed a significantly worse 5‐year graft survival rate (77.3%; vs. DSA‐negative, 91.6%). Six of 14 LCT‐positive patients exhibited graft loss shortly after transplantation (5‐year survival rate: 57.1%). All LCT‐positive patients with graft loss underwent left lobe living donor liver transplantation. Significantly lower ratio of graft volume relative to standard liver volume (32.9 ± 5.7%) and smaller graft size (365.3 ± 57.9 g) were observed in patients with graft loss (p < .03, vs. surviving grafts). Significantly higher DSA‐mean fluorescence intensity (MFI) values were present in patients with graft loss (p = .0012, vs. surviving grafts). Conclusions Patients with preformed DSAs exhibited worse graft outcomes in living donor liver transplantation. Higher DSA‐MFI values and smaller graft size were associated with worse outcomes in LCT‐positive patients. High‐risk patients with preformed DSAs should be considered for appropriate graft selection and application of a desensitization protocol.

Immunological behavior of graft-infiltrating lymphocytes (GILs) determines the graft fate (i.e., rejection or acceptance). Nevertheless, the functional alloreactivity and the phenotype of GILs at various times during the early post-transplantation phase have not been fully elucidated. We examined the immunological activities of early-phase GILs using a murine model of cardiac transplantation. GILs from 120-h allografts, but not 72-h allografts, showed robust activation and produced proinflammatory cytokines. In particular, a significant increase in CD69 + T-bet + Nur77 + T cells was detected in 120-h allografts. Furthermore, isolated GILs were used to reconstitute BALB/c Rag2 À/À cc À/À (BRG) mice. BRG mice reconstituted with 120-h GILs displayed donor-specific immune reactivity and rejected donor strain cardiac allografts; conversely, 72-h GILs exhibited weak antidonor reactivity and did not reject allografts. These findings were confirmed by re-transplantation of cardiac allografts into BRG mice at 72-h post-transplantation. Re-transplanted allografts continued to function for >100 days, despite the presence of CD3 + GILs. In conclusion, the immunological behavior of GILs considerably differs over time during the early post-transplantation phase. A better understanding of the functional role of early-phase GILs may clarify the fate determination process in the graftsite microenvironment.

In organ transplantation, a reproducible and robust immune-monitoring assay has not been established to determine individually tailored immunosuppressants (IS). We applied humanized mice reconstituted with human (hu-) peripheral blood mononuclear cells (PBMCs) obtained from living donor liver transplant recipients to evaluate their immune status. Engraftment of 2.5 × 10 6 hu-PBMCs from healthy volunteers and recipients in the NSG mice was achieved successfully. The reconstituted lymphocytes consisted mainly of hu-CD3 + lymphocytes with predominant CD45RA -CD62L lo TEM and CCR6 -CXCR3 + CD4 + Th1 cells in hu-PBMC-NSG mice. Interestingly, T cell allo-reactivity of hu-PBMC-NSG mice was amplified significantly compared with that of freshly isolated PBMCs (p<0.05). Furthermore, magnified hu-T cell responses to donor antigens (Ag) were observed in 2/10 immunosuppressed recipients with multiple acute rejection (AR) experiences, suggesting that the immunological assay in hu-PBMC-NSG mice revealed hidden risks of allograft rejection by IS. Furthermore, donor Ag-specific hyporesponsiveness was maintained in recipients who had been completely weaned off IS (n=4), despite homeostatic proliferation of hu-T cells in the hu-PBMC-NSG mice. The immunological assay in humanized mice provides a new tool to assess recipient immunity in the absence of IS and explore the underlying mechanisms to maintaining operational tolerance.

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