中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (12): 2161-2165.doi: 10.3969/j.issn.1673-8225.2011.12.018

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壳聚糖球形多孔微载体支持下人肝细胞L-02的高浓度细胞培养

张  瑞1，韩宝三2，彭承宏2   

  1. 1内蒙古医学院第一附属医院肿瘤外科，内蒙古自治区呼和浩特市  010059
    2上海交通大学医学院附属瑞金医院器官移植中心，上海市 200025
  • 收稿日期:2010-11-24 修回日期:2011-01-06 出版日期:2011-03-19 发布日期:2011-03-19
  • 作者简介:张瑞☆，男，1972年生，内蒙古自治区呼和浩特市人，汉族，上海交通大学医学院附属瑞金医院毕业，博士，副主任医师，主要从事肿瘤外科的临床和基础研究。 ruizhang999@sohu.com
  • 基金资助:

    国家高新技术研究发展计划“863”项目（2007AA02Z487），课题名称：微粒生物人工肝反应器构建的研究；国家自然科学基金项目(30672043)，课题名称：微粒肝细胞生物反应器－微囊化三维多孔微载体构建的研究；国家自然科学基金项目(30772105)，课题名称：新型超分子自组装微囊/肝细胞球形聚集体构建的研究；国家自然科学基金项目(20074031)，课题名称：人肝细胞/聚合物杂化界面和生物人工肝的研究。

Culture of human hepatocyte L-02 at high concentration based on spherical porous chitosan microcarriers

Zhang Rui1, Han Bao-san2, Peng Cheng-hong2   

  1. 1Department of Oncosurgery, First Affiliated Hospital, Inner Mongolia Medical College, Hohhot  010059, Inner Mongolia Autonomous Region, China
    2Organ Transplantation Center, Rujin Hospital Affiliated to Medical College of Shanghai Jiao Tong University, Shanghai  200025, China
  • Received:2010-11-24 Revised:2011-01-06 Online:2011-03-19 Published:2011-03-19
  • About author:Zhang Rui, Doctor, Associate chief physician, Department of Oncosurgery, First Affiliated Hospital, Inner Mongolia Medical College, Hohhot 010059, Inner Mongolia Autonomous Region, China ruizhang999@sohu.com
  • Supported by:

    the National High-tech Research and Development Plan of China - the "863" Research Project, No. 2007AA02Z487*; the National Natural Science Foundation of China, No. 20074031*, 30672043* , 30772105*

PDF

319

被引次数

4

摘要：

背景：微载体培养技术作为一项体外高浓度细胞培养技术，近年来已在肝细胞的体外培养中得到应用。
目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。
方法：以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组；无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数，并对实验组进行形态学观察，包括倒置相差生物显微镜观察和扫描电子显微镜观察。
结果：两组培养的细胞数量均呈现前3 d增长，在第3天细胞数量达到最高值；而且实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P < 0.05)，实验组各样本之间差异无显著性意义(P > 0.05)；倒置相差生物显微镜下动态观察，可见前3 d微载体表面黏附生长的肝细胞则逐渐增多，第3天可见大部分微载体表面有许多肝细胞黏附成团，总的存活率均在90%以上，且肝细胞保持着良好的形态学结构；扫描电子显微镜观察，微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。提示，以自制的壳聚糖球形多孔微载体作为一种支架，在体外三维环境下可以进行高浓度细胞培养。

关键词: 微载体, 壳聚糖, 人工肝脏, 肝细胞培养, 组织工程

Abstract:

BACKGROUND: Recently, microcarrier culture as a high concentration cell culture technology has been used in hepatocyte culture in vitro.
OBJECTIVE: To periodically observe the morphological changes of human hepatocyte L-02 cultured by the spherical porous chitosan microcarrier.
METHODES: The self-made spherical porous chitosan microcarrier sample as a support to culture human hepatocyte L-02 as experimental group; non-spherical porous chitosan microcarrier as a support to culture human hepatocyte L-02 as control group. Cells in two groups underwent cell count, and the morphological changes were observed in experimental group, including inverted phase contrast biological microscope observation and scanning electron microscope observation.
RESULTS AND CONCLUSION: The quantity of the two groups’ cultured cells was increased in the former 3 days and reached the highest value at the third day. The cell quantity in experiment group was obviously higher than the number of cells without microcarrier culture in control group (P < 0.05). There was no significant difference of samples in experimental group (P > 0.05). The quantity of hepatocytes adhered to microcarrier surface was gradually increased in the former 3 days under inverted phase contrast biological microscope dynamic observation. There were lots of hepatocytes adhered to ball in the surface of a greater part of microcarrier, the total survive rate of hepatocyte was more than 90%, and hepatocyte kept a good morphological structure. Under scanning electron microscope, lots of hepatocytes adhered intensely each other in the surface, section, and inside of microcarrier. It is indicated that the self-made spherical porous chitosan microcarrier as a support in three-dimensional environment can undergo cell culture with high concentration.

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