动植物细胞工.程.pptVIP

4.1 动物细胞特点 无细胞壁 倍增时间长，生长缓慢 需氧量少，对搅拌敏感 聚集体形成 原代细胞培养50代即开始退化 4.2 动物细胞培养定义 动物细胞与组织培养是从动物体内取出细胞或者组织，模拟体内的生理环境，在无菌、适温和丰富的营养条件下，使离体细胞或者组织生存、生长并维持结构和功能的一门技术。 是动物细胞工程的基础。 4.3 发展历史 1885年W Roux 鸡胚神经板 1907，美国的哈里森使用盖玻片悬滴培养蛙胚神经组织细胞 A Carrel 无菌技术 1951 W Earle 培养基 。。。 4.4 生长特性 贴附生长型 如人胚肺细胞，Hela细胞，巨噬细胞，神经细胞 上皮样、成纤维样、无定形 单层、汇合 悬浮生长型细胞 如血液白细胞，淋巴组织细胞等 常用术语 Cell, tissue organ culture 贴壁依赖性、汇合 原/初代培养Primary culture 传代 细胞系（有限、无限/连续）[已建成的] 二倍体、转化 细胞株（有限、无限/连续） 克隆 接触抑制 4.5 体外培养细胞基本技术 体外培养特点 体外培养工具 体外培养条件 体外细胞生长增殖过程 培养方法 大规模培养技术 4.5.1 体外培养特点 营养条件苛刻 适应性差, 敏感 培养时间长，易污染 Animal cells are more difficult to culture than microorganisms because they require many more nutrients and typically grow only when attached to specially coated surfaces. Despite these difficulties, various types of animal cells, including both undifferentiated and differentiated ones, can be cultured successfully. 4.5.2 体外培养工具 空心纤维 微球 WORK ENVIRONMENT AND SURFACE Laminar flow hood Relatively enclosed space Little traffic flowing past work space Confined space can be easily cleaned and maintained Must have annual checkup of HEPA filters Should have internal outlets for electricity, vacuum and gas Frequently outfitted with UV light Prevent contamination by daily scrub with 70% ethanol Use closed flask attached to vacuum for spent medium Separate sterile room All incoming air circulated through HEPA filters PLASTICWARE AND GLASSWARE Pipettes, bottles, flasks, petri dishes ? Plastic -- better attachment and cell growth of monolayer cell culture -- more expensive than reusable glass -- suitable for storage at 4oC ? Glass -- Can withstand temperatures 0oC use for maintaining frozen stocks -- Steam sterilize by autoclaving * relies on steam pressure and high heat * use fast-dry cycle to dry condensate * use “wet” cycle for salt solutions toprevent evaporation * loosely place caps on bottles 4.5.3 体外培养条件 温度，37度 pH：7.2-7.4 气体，氧，二氧化碳，氮气 营养条件（天然/合成培养基） 需要多种氨基酸，维