Kuge Hiroyuki, Ohashi Kazuo, Yokoyama Takashi, Kanehiro Hiromichi, Hisanaga Michiyoshi, Koyama Fumikazu, Bumgardner Ginny L, Kosai Ken-Ichiro, Nakajima Yoshiyuki
Department of Surgery, Nara Medical University, Nara, Japan.
Cell Transplant. 2006;15(1):1-12.
Cell-based therapies, including liver tissue engineering following hepatocyte transplantation, have therapeutic potential for several types of liver diseases. Modifications in the methodology to manipulate the donor hepatocytes in a more simple and timely manner prior to transplantation would enhance the therapeutic efficacy of this procedure. Conventional approach for vector-mediated gene transduction to the isolated hepatocytes has been performed under primary culture conditions that routinely require several days to complete. In our study, we have established a clinically feasible approach that requires only 1 h of infection time with an adenoviral vector system that results in an extremely efficient transduction efficiency (> 80%). To optimize transduction efficiency and sustain normal cellular function, we determined that the isolated hepatocytes should be maintained in UW solution as a suspension medium and infected with adenoviral vectors (Ad) for no more than 1 h at a MOI of 1. To establish if the isolated hepatocytes could be used as a source for cell-based therapies, we transplanted the Ad-transduced hepatocytes into the liver or under the kidney capsule. When the cells were transplanted into the liver, Ad-transduced hepatocytes cultured in suspension conditions were found to have a significantly higher survival rate (p < 0.01) than Ad-transduced hepatocytes cultured under standard conditions. We also confirmed that these Ad-transduced hepatocytes have ability to survive long term and were able to engineer a biologically active hepatic tissue under the kidney capsule. Finally, we obtained high level of transduction into canine, porcine, and human isolated hepatocytes in a suspension solution mixed with Ad. In conclusion, the present studies demonstrate that isolated hepatocytes could be genetically modified using Ad when kept in a suspension solution. For this reason, this cell-modified technique could be used for the treatment of liver-targeted diseases and/or disorders.
基于细胞的疗法，包括肝细胞移植后的肝组织工程，对多种类型的肝脏疾病具有治疗潜力。在移植前以更简单、及时的方式操纵供体肝细胞的方法改进，将提高该程序的治疗效果。将载体介导的基因转导至分离的肝细胞的传统方法是在原代培养条件下进行的，这通常需要几天时间才能完成。在我们的研究中，我们建立了一种临床可行的方法，该方法使用腺病毒载体系统仅需1小时的感染时间，就能产生极高的转导效率（>80%）。为了优化转导效率并维持正常细胞功能，我们确定分离的肝细胞应保存在UW溶液中作为悬浮培养基，并以1的感染复数用腺病毒载体（Ad）感染不超过1小时。为了确定分离的肝细胞是否可作为基于细胞的疗法的来源，我们将Ad转导的肝细胞移植到肝脏或肾包膜下。当细胞被移植到肝脏中时，发现悬浮条件下培养的Ad转导肝细胞的存活率显著高于标准条件下培养的Ad转导肝细胞（p<0.01）。我们还证实，这些Ad转导的肝细胞具有长期存活的能力，并且能够在肾包膜下构建有生物活性的肝组织。最后，我们在与Ad混合的悬浮溶液中实现了对犬、猪和人分离肝细胞的高效转导。总之，本研究表明，分离的肝细胞在悬浮溶液中保存时可用Ad进行基因修饰。因此，这种细胞修饰技术可用于治疗肝脏靶向疾病和/或病症。
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