中国组织工程研究与临床康复2008，Vol.12Issue(45)：8963-8968,6.
体外培养和冷冻保存对微囊化细胞生长和内皮抑素表达的影响
Effects of in vitro culture and cryopreservation on microencapsulated recombinant cell growth and endostatin production
摘要
Abstract
BACKGROUND: Transplantation of microencapsulated recombinant cells needs large amount of biological microcapsules with high cell viability and protein expression. In vitro culture and cryopreservation are two important processes in microcapsule preparation.OBJECTIVE: To explore the effects of in vitro culture time and cryopreservation on microencapsulated recombinant Chinese hamster ovary (CHO) cell growth in vivo and endostatin expression and the effects of in vitro culture time on cryopreservation of microencapsulated cells.DESIGN, TIME AND SETTING: The experiment was performed at the Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences from April to October 2006.MATERIALS: Alginate and Poly-L-lysine was purchased from Sigma, USA.METHODS: The microcapsules containing recombinant CHO cells were inoculated into a 24-well plate. Each well contained 0.1 mL microcapsules and 2 mL DMEM/F12 medium. The cells were incubated in 5% CO<,2> at 37 ℃. Viable cell density in microcapsule was measured by MTT every 2 days, and the supernatant was collected to determine the concentration of endostatin.MAIN OUTCOME MEASURES: Growth of microencapsulated cell and endostatin expression.RESULTS: In vitro culture time greatly affected the in vivo growth of the microencapsulated cells, endostatin expression and stability of the microcapsules, Microencapsulated recombinant CHO cells grew well without in vitro culture or in vitro cultured 4 days, and endostatin expression kept high. However, the microencapsulated recombinant CHO cells cultured in vitro for 8 days ruptured on day 26 post-transplantation. In addition, in vitro culture time also had effect on cryopreservation of the microencapsulated cells. The microencapsulated cells cultured in vitro for 4 days and for 8 days grew well and had high endostatin expression after cryopreserved in liquid nitrogen for 40 days. On the contrary, the microencapsulated recombinant CHO cells without in vitro culture before cryopreservation were almost all dead after thawing.CONCLUSION: The appropriate in vitro culture time for biological microcapsules is 4 days. Cryopreservation has no significant effect on in vivo growth of microencapsulated cells, endostatin expression and stability of the microcapsules.关键词
细胞移植/体外培养时间/冷冻保存/重组内皮抑素细胞/内皮抑素分类
医药卫生引用本文复制引用
张英,王为,吕国军,于炜婷,郭昕,雄鹰,马小军..体外培养和冷冻保存对微囊化细胞生长和内皮抑素表达的影响[J].中国组织工程研究与临床康复,2008,12(45):8963-8968,6.基金项目
国家"九七三"攻关项目资助:细胞的物理化学修饰(2002CB713804),组织工程学重要基础科学问题研究-组织微环境对干细胞形成特定组织的影响及其作用机制(2005CB522702).国家自然科学基金资助:微环境中细胞生长与生产的特性与基本规律(20236040),长期持续分泌型生物技术药物释放系统的研究(30472102)Supported by: theNational Basic ResearchProgram of China, No. 2002CB713804, 2005CB522702 （2002CB713804）
the National Natural Science Foundation of China, No. 20236040, 30472102 （）
