Kobayashi N, Taguchi T, Noguchi H, Okitsu T, Totsugawa T, Watanabe T, Matsumura T, Fujiwara T, Urata H, Kishimoto N, Hayashi N, Nakaji S, Murakami T, Tanaka N
First Department of Surgery, Okayama University Medical School, Japan.
Cell Transplant. 2001;10(4-5):387-92.
With the development of biotechnology, hepatic support by a hybrid artificial liver (HAL) using hepatocytes has been given much attention. Because the availability of human livers is limited, we have established a tightly regulated immortal human hepatocyte cell line, NKNT-3, for developing HAL. Because high-density cell culture allows the compactness of the HAL device and its easy use under emergency circumstances, we have developed cell adhesive GRGDS peptide-containing cellulose microspheres (GRGDS/CMS). The GRGDS/CMS efficiently immobilized NKNT-3 cells within 24 h in a stirred suspension culture. Electron microscopic examinations demonstrated glycogen granules and well-developed endoplasmic reticulum and mitochondria in NKNT-3 cells attached to the GRGDS/CMS. The cells showed ammonia clearance activity, whereas HepG2-transformed human liver cells did not remove the loaded ammonia. An efficient adenoviral delivery of the lacZ reporter gene was performed in GRGDS/CMS-immobilized NKNT-3 cells. In this study we present rapid immobilization of NKNT-3 immortal human hepatocytes using cellulose microspheres carrying GRGDS peptides. These microspheres satisfied immediate preparation of NKNT-3 cells in sufficient quantity and of adequate quality.
随着生物技术的发展，利用肝细胞的混合人工肝（HAL）进行肝脏支持受到了广泛关注。由于人类肝脏的可用性有限，我们建立了一种严格调控的永生化人肝细胞系NKNT-3，用于开发HAL。由于高密度细胞培养可使HAL装置紧凑且便于在紧急情况下使用，我们开发了含细胞黏附性GRGDS肽的纤维素微球（GRGDS/CMS）。在搅拌悬浮培养中，GRGDS/CMS能在24小时内有效固定NKNT-3细胞。电子显微镜检查显示，附着在GRGDS/CMS上的NKNT-3细胞内有糖原颗粒、发达的内质网和线粒体。这些细胞具有氨清除活性，而经HepG2转化的人肝细胞则不能去除加载的氨。在GRGDS/CMS固定的NKNT-3细胞中进行了lacZ报告基因的高效腺病毒递送。在本研究中，我们展示了使用携带GRGDS肽的纤维素微球快速固定NKNT-3永生化人肝细胞的方法。这些微球满足了立即制备足够数量和质量的NKNT-3细胞的需求。
