The Optimization of the Hepatocyte Surface Modification Procedures in Terms of Heparin and Apyrase for Improving Hepatocyte Engraftment
Hiroyuki Ogasawara1, Yuji Teramura2,3, Takehiro Imura4, Akiko Inagaki4, Yoshikatsu Saito1, Muneyuki Matsumura1, Kengo Fukuoka1, Ibrahim Fathi4, Shigehito Miyagi1, Kristina Nilsson3, Kazuo Ohashi5, Michiaki Unno1, Takashi Kamei1, Susumu Satomi1, Bo Nilsson3, Elisabet Gustafson6, Masafumi Goto1,4.
1Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan; 2Department of Bioengineering, The University of Tokyo, Tokyo, Japan; 3Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Uppsala, Sweden; 4Division of Transplantation and Regenerative Medicine, Tohoku University, Sendai, Japan; 5Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; 6Department of Women's and Children's health, Division of Pediatric Surgery, Uppsala University, Uppsala, Sweden
Background: The 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.
Methods: Rat 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.
Results: Regarding 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.
Conclusions: The 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.
