The Optimization of the Hepatocyte Surface Modification Procedures in Terms of Heparin and Apyrase for Improving Hepatocyte Engraftment


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Abstract

BackgroundThe rapid destruction of hepatocyte grafts immediately after transplantation has hampered the application of this procedure as a clinical treatment. The instant blood-mediated inflammatory reaction (IBMIR), which is characterized by the activation of both coagulation and complement cascades, is a plausible underlying cause for this poor engraftment. In pancreatic islet transplantation, which shares many aspects with hepatocyte transplantation, it has been reported that islet surface heparinization efficiently inhibits the IBMIR without increasing the bleeding risk. In the present study, we tried to optimize the hepatocyte surface modification procedures in terms of heparin and apyrase for improving hepatocyte engraftment.MethodsRat hepatocytes were isolated using a modified two-step collagenase perfusion technique and then purified by Percoll density gradient centrifugation. Hepatocytes (2.0 × 106) were mixed with heparin-binding peptide (CNSAHRTRGRQRS)-poly (ethylene glycol)-conjugated phospholipid (HBP-PEG-lipid) solution or Maleimide-PEG-lipid solution, and then a solution of heparin conjugate (Corline AB, Uppsala, Sweden) or apyrase-SH was added for 10 minutes. All of these procedures were performed at either room temperature (RT) or 4 °C using either phosphate-buffered saline (PBS) or optimized culture medium for hepatocytes with or without fetal bovine serum (FBS). The hepatocyte viability was evaluated by a trypan blue exclusion assay. The efficiency of the hepatocyte surface modification procedures was evaluated by detecting AlexaFluor 488 under fluorescence microscopy.ResultsRegarding both heparin and apyrase, the surfaces of hepatocytes were sufficiently coated in all groups. The hepatocyte viability prior to the surface modification procedures was 94.09±2.18%. When PBS was used for the procedures, the viability of heparin-coated hepatocytes was 26.78±5.27% at RT and 63.53±5.95% at 4 °C. Likewise, the viability of apyrase-coated hepatocytes was 33.04±11.37% at RT and 60.43±6.63% at 4 °C. Of particular note, the viability of non-coated hepatocytes was 57.03±17.25% at RT and 66.65±1.28% at 4 °C, suggesting that the hepatocyte viability was substantially damaged during the preservation period rather than due to the coating chemicals per se. When using optimized culture medium for hepatocytes at 4 °C, the viability of heparin-coated and apyrase-coated hepatocytes markedly increased to 85.76±0.74% and 85.96±3.51%, respectively, under the supplementation of FBS, whereas the viability of heparin-coated hepatocytes remained at 73.79±7.42% without FBS.ConclusionsThe present study revealed that hepatocyte surface modification in terms of heparin and apyrase could be efficiently performed with minimal damage to the hepatocytes by introducing an optimized culture medium for hepatocytes including FBS, instead of carrying out the standard procedure (PBS), at 4 °C.

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